Albumin Variants And Conjugates

Abstract

Based on the three-dimensional structure of albumin, the inventors have designed variant polypeptides (muteins) which have one or more cysteine residues with a free thiol group (hereinafter referred to as "thio-albumin"). The variant polypeptide may be conjugated through the sulphur atom of the cysteine residue to a conjugation partner such as a bioactive compound.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of U.S. patent application Ser. No. 13/201,123 filed Aug. 11, 2011, pending, which is a 35 U.S.C. 371 national application of PCT/EP2010/051751 filed Feb. 11, 2010, which claims the benefit of priority to European application nos. 09152625.1 and 09152686.3, filed Feb. 11, 2009 and Feb. 12, 2009, respectively, and U.S. provisional application No. 61/154,555, filed Feb. 23, 2009. The contents of each of U.S. patent application Ser. No. 13/201,123, PCT application no. PCT/EP10/51751, European application no. 09152625.1, European application no. 09152686.3, and U.S. provisional application No. 61/154,555 are fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

[0003] The present invention relates to conjugation competent albumins and albumin-related polypeptides, and their conjugates with at least one moiety, and to polynucleotides encoding them.

BACKGROUND OF THE INVENTION

[0004] Serum albumins provide valuable scaffolds to which bioactive molecules may be fused, either through genetic fusions or chemical fusions to improve the properties of the fused molecule(s) (Leger, R. et al. (2004). Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau, K., et al. (2005). Bioconjug Chem 16(4): 1000-8; Balan, V. et al. (2006). Antivir Ther 11(1): 35-45; EP 0 413 622; WO 90/13653; EP 1 681 304; WO 1997/024445; WO 01/79271). Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused bioactive molecule from inactivation.

[0005] The biochemistry, genetics and medical applications of albumins are well characterized ("All about Albumin", T. Peters Jr., Academic Press N.Y., and http://www.albumin.org/). Human serum albumin (HSA, also referred to as HA) is the most abundant protein in human plasma at .about.60 g/L. The sequence of HSA is provided in SEQ ID No. 1. Natural variants of HSA occur and a list of know polymorphisms is given in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and at http://www.uniprot.org/uniprot/P02768.

[0006] The production and purification of recombinant human albumins are well established (WO 95/23857; WO 00/44772; WO 2006/066595; EP 0 305 216; Sleep et al. 1990 Biotechnology (NY). 1990 January; 8(1):42-6)) and include recombinant human albumin for pharmaceutical applications (Bosse et al. (2005). J Clin Pharmacol 45(1): 57-67). The three-dimensional structure of HSA has been elucidated by X-ray crystallography (Carter et al. (1989). Science 244(4909): 1195-8; Sugio et al. (1999). Protein Eng 12(6): 439-46). The HSA polypeptide chain has 35 cysteine residues, which form 17 disulphide bonds and one unpaired (free) cysteine at position 34 of the mature protein (Seq ID No. 1). Cysteine-34 has been used to for conjugation of molecules to albumin (Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8), and provides a precise, well defined site for conjugation. However, conjugation at cysteine-34 provides only one site for attachment of a single moiety thus there is no choice of conjugation site. Also, the provision of a single conjugation sites means that only one moiety can be conjugated to each albumin molecule. What is required is an albumin molecule which provides one or more alternative attachment sites.

SUMMARY OF THE INVENTION

[0007] Based on an analysis of the three-dimensional structure of a human serum albumin (HSA), conserved residues within albumin polypeptides and natural polymorphisms thereof, the inventors have designed variant polypeptides (muteins) of albumin which have one or more conjugation competent cysteine residues. The term `thio-albumin` is used herein to describe an albumin variant which comprises one or more unpaired cysteine residues, particularly an albumin variant in which one or more of the unpaired cysteine residues does not occur in a naturally occurring variant of an albumin. Thus a thio-albumin is a `conjugation competent albumin`. A thio-albumin may be referred to as a `cysteine variant of an albumin`.

[0008] Throughout this specification, the term `albumin` includes naturally occurring albumin, albumin-related proteins and variants thereof such as natural and engineered variants. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins. The albumin may have at least 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99% similarity or identity to SEQ ID No. 1. Thus a thio-albumin of the invention may be a derivative of, or be based on, any of such albumin.

[0009] The unpaired cysteine residues may be provided by insertion, deletion, substitution, addition or extension of an albumin sequence.

[0010] The invention also relates to a conjugate comprising at least one, for example 2, 3, 4, 5 or 6, conjugation partners such as bioactive compounds and a polypeptide according to the invention

[0011] The invention also provides a method for designing conjugation-competent albumins.

BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1. is a table showing criteria used to select sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of conjugation competent cysteines.

[0013] FIG. 2. is an alignment of the amino acid sequence of human serum albumin (SEQ ID No. 1=Human-P02768.pro) with albumins from fifteen other mammalian species. `Majority` shows the consensus sequence. `+ Majority` shows the relative homology between all sixteen sequences in bar chart form, where the height of the bar indicates the relative homology at 20, 40, 60, 80 and 100%. The protein sequences include the leader sequence.

[0014] FIG. 3. Is an alignment of the amino acid sequence of human serum albumin (P02768.pro=SEQ ID No. 1) with albumins from thirty two other species, some of which are mammalian. `Majority` shows the consensus sequence. `+ Majority` shows the relative homology between all thirty three sequences in bar chart form, where the height of the bar indicates the relative homology at 20, 40, 60, 80 and 100%. The protein sequences include the leader sequence. P02768.pro: human; P02769.pro: bovine; P49064.pro: cat; P49822.pro: dog; Q5XLE4.pro: donkey; JC5838.pro: gerbil; ACF10391,1.pro: goat fragment; AAQ20088.pro: guinea pig; P35747.pro: horse; Q28522.pro: macaque; P07724.pro: mouse; P08835.pro: pig; P02770.pro: rat; P49065.pro: rabbit; Q28522.pro: Rhesus monkey; P14639.pro: sheep; NP_001127106.pro: orangutan; P19121.pro: chicken; P01012.pro: chicken ovalbumin; O73860.pro: turkey ovalbumin; AAC63407.pro: sea lamprey; Q91274.pro: sea lamprey; P21847.pro: bullfrog; AAD09358.pro: Rana shqiperica; ABXL68.pro: Xenopus; NP_001004887.pro: Xenopus; AAL56646.pro: Spotted Salamander; Q03156.pro: Atlantic salmon; P21848.pro: Atlantic salmon; AAM46104.pro: Sphenodon punctatus; P83517.pro: Australian lungfish; S59517.pro: monocled cobra; AAL08579.pro: Schistosoma mansoni.

[0015] FIG. 4. is a Venn diagram showing the classes of and relationship between twenty amino acids.

[0016] FIGS. 5A, 5B, 5C and 5D are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for amino acid substitutions, insertions and deletions for the generation of one or more conjugation competent cysteine.

[0017] FIGS. 6A and 6B are tables showing groups of preferred sites in human serum albumin (SEQ ID No. 1) for disruption of one or more disulphide bonds for the generation of one or more conjugation competent cysteines.

[0018] FIG. 7. is a map of plasmid pDB2244.

[0019] FIG. 8 is a map of plasmid pDB2243.

[0020] FIG. 9. is a map of plasmid pDB2713.

[0021] FIG. 10 is a table showing preferred sites for conjugation grouped according to their relative position on a folded albumin of SEQ ID No. 1.

[0022] FIG. 11 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having a single free thiol group in addition to Cys-34 of native human serum albumin.

[0023] FIG. 12 is a map of plasmid pDB3927.

[0024] FIG. 13 is a map of plasmid pDB3964.

[0025] FIG. 14 is a map of plasmid pBD3936.

[0026] FIG. 15 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation) of albumin molecules having a free thiol group at Cys-34 of SEQ ID No. 1 and an additional free-thiol at the position indicated below the bar chart.

[0027] FIG. 16 is a table showing mutations (see second column) made to native human serum albumin to generate molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed.

[0028] FIG. 17 shows SDS-PAGE analysis, and HPLC data (bar chart), showing the expression (pg/ml with standard deviation of albumin molecules having one or more free thiol groups in addition to Cys-34 of native human serum albumin and/or having Cys-34 removed.

[0029] FIG. 18 is a table showing the fermentation yield and relative level of conjugation to albumin molecules comprising one or more free-thiols.

[0030] FIG. 19 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) before treatment with DTNB.

[0031] FIG. 20 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and L585C) after treatment with DTNB.

[0032] FIG. 21 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) before treatment with DTNB.

[0033] FIG. 22 is a mass spectrogram of a rHA molecule designed to have four free-thiols (Cys-34, D129C, C360S and L585C) after treatment with DTNB.

[0034] FIG. 23 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol) before treatment with DTNB.

[0035] FIG. 24 is a mass spectrogram of a rHA molecule designed to have three free-thiols (Cys-34, A2C and a C-terminal free-thiol).

DETAILED DESCRIPTION OF THE INVENTION

[0036] A first aspect of the invention provides a method for designing and/or preparing variant albumins comprising one or more conjugation competent cysteine residues. Therefore, the polypeptide may be considered to be conjugation-competent. Such an albumin may be referred to as a `thio-albumin` or as a `cysteine varant` of an albumin. The term `conjugation competent cysteine` includes a cysteine which has a thiol which is not disulphide bonded to another cysteine and which is, preferably, not blocked from conjugating to another molecule (which may be referred to as a `conjugation partner`) due to unfavourable steric hindrances. That is, preferably the location of the cysteine within or on a folded polypeptide is such that it is available for conjugation.

[0037] A number of selection criteria may or may not be used alone or in any combination in order to identify suitable sites for introduction of a conjugation competent cysteine residue. Therefore, the invention provides a method and/or rules for a priori identification of sites of an amino acid sequence of albumin at which a conjugation competent cysteine may be introduced. Such sites may be referred to as `candidate residues`. The albumin sequence on which the variant albumin is based may be SEQ ID No. 1 or any other albumin. For example, the variant albumin may be be based on an albumin which does or does not have a cysteine at position 34 of the amino acid sequence, or an equivalent position. Cysteine residues may or may not be introduced by one or more of substitution, insertion, deletion, extension and addition. Sites may or may not be selected with reference to a 3-dimensional structure of an albumin or variant thereof. The following criteria may or may not be used to select suitable sites:

[0038] (a) Solvent Accessible Surface Area ("Surface Accessibility" (% SASA)).

[0039] Preferably the surface accessibility is high. For example, preferably the surface accessibility is at least 60%, more preferably, from 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% to 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100%. % SASA may be determined as a `raw score` using the methods described herein or may be calculated relative to the score of the residue which has the maximum surface accessibility in the protein. For example, the albumin of HSA 1AO6 has a maximum surface accessibility of 229.0 and this is the highest scoring residue in HSA. A higher surface accessibility indicates that the residue is on the surface of the protein and is therefore available for binding. Such accessibility may be calculated using a method as described herein.

[0040] (b) Presence or Absence of Crystallographic B-Factor(s).

[0041] B-factor indicates relative flexibility of an amino acid residue within a 3-dimensional structure. Preferably the B-factor is from at least 30, 40, 50, 60, 70, 80 or 90% to at least 40, 50, 60, 70, 80, 90 or 100% which may or may not be relative to the maximal B-factor score of any amino acid residue within the molecule. For HSA (e.g. 1AO6), preferably the B-factor score is high, for example from at least 30, 40, 50, 60, 70, 80, 90, or 100 to at least 40, 50, 60, 70, 80, 90, 100 or 106 (for example using the B-factor scoring system described herein). Alternatively the B-factor score may be less than or equal to 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10%, as described herein.

[0042] The B-Factor (root mean square fluctuations) of the C-alpha carbon atoms during the last nanosecond of the simulation may be calculated using the Gromacs tool "g_rmsf", version 3.3, based on D. van der Spoel, E. Lindahl, B. Hess, G. Groenhof, A. E. Mark and H. J. C. Berendsen: GROMACS: Fast, Flexible and Free, J. Comp. Chem. 26 pp. 1701-1718 (2005).

[0043] (c) Presence of Absence of Secondary Structure (SS).

[0044] The candidate residue may or may not be located within secondary structure for example H (Helix), B (isolated beta bridge) or E (Extended sheet). Location of the residue outside of secondary structure indicates that the residue is less likely to be important to secondary structure and/or is more likely to be available for binding than a residue located within secondary structure.

[0045] (d) Relative Homology with Other Albumins.

[0046] Within a given protein sequence, an amino acid residue showing high homology with other similar sequences is likely to indicate such a residue or region is likely to be important to the structure and/or function of the protein. Therefore it is preferred that a candidate residue shows a homology of less than 100% relative to alignment of the albumin in which the residue is located with known albumins (e.g. mammalian albumins such as those shown in FIG. 2 or a combination of mammalian and non-mammalian albumins such as those shown in FIG. 3). A homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 is preferred. Homology can be determined using algorithms known in the art such as Clustal, e.g. Clustal W (Thompson et al. (1994). Nucleic Acids Res 22(22): 4673-80) or Clustal V (Higgins, D. G. and P. M. Sharp (1989). "Fast and sensitive multiple sequence alignments on a microcomputer." Comput Appl Biosci 5(2): 151-3.). Lower homology indicates that the residue is not particularly important or critical to the structure and/or function of the protein. Preferably the homology is determined with reference to the sixteen mammalian albumins of FIG. 2 or the thirty three mammalian and non-mammalian albumins of FIG. 3.

[0047] (e) Presence or Absence of Adjacent Conserved Residues.

[0048] Within an amino acid sequence, each residue has one or two adjacent residues. If a candidate residue is immediately adjacent one or more residues having a low homology, relative to known albumins, this indicates that the candidate residue is unlikely to be particularly important or critical to the structure and/or function of the protein. This is because the candidate residue is likely to be located within a relatively unconserved region of the protein. It is therefore preferred that the candidate residue is not adjacent a residue which has 100% homology relative to alignment of the albumin with known albumins. Homology may be determined as described herein. The candidate residue may be adjacent two residues (i.e. one residue C-terminal relative to the candidate residue and one residue N-terminal relative to the candidate residue) which each have 100% homology relative to alignment of the albumin with known albumins (e.g. FIG. 2 or FIG. 3). It is preferred that the candidate residue is adjacent one or two residues having a homology of less than 100, 98, 96, 95, 94, 92, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5--these levels of homology may be referred to as `thresholds`. Homology may be determined as described herein. Taking into account the homology threshold, the location of an amino acid relative to a conserved region may be quantified, for example by scoring an amino acid which is not adjacent any amino acid exceeding the homology threshold as 0, scoring an amino acid which is adjacent one amino acid exceeding the threshold as 1 and scoring an amino acid which adjacent two amino acids exceeding the threshold as 2.

[0049] (f) Evidence for Polymorphism(s).

[0050] A polymorphism is a genetic variation, a polymorphism may or may not cause a phenotypic change to the resultant protein. Preferably the candidate residue is not at a position for which a polymorphism causing a phenotypic change is known. More preferably, the candidate residue is not at a position for which a polymorphism causes, or is known to cause, thermal instability. Polymorphisms known for HSA (SEQ ID No. 1) are detailed in FIG. 1 and are also discussed in Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16 and at http://www.uniprot.org/uniprot/P02768. The presence, absence and/or effect of a polymorphism may be quantified, for example by scoring a known polymorphism that has no phenotypic change as 0, scoring a polymorphism where a phenotypic change is known (but not known to cause thermal instability) as 1 and scoring a polymorphism which is known to cause thermal instability as 2.

[0051] (g) Relative Conservation of Candidate Amino Acid and Cysteine.

[0052] Amino acids fall into various well known classes. Therefore, some amino acids are more closely related than others. The introduced cysteine residue may or may not maintain a relatively high level of conservation with the candidate amino acid. FIG. 4 is a Venn diagram which provides one system by which conservation level can be quantified. The scoring system of FIG. 4 uses a scale of 0 to 5 in which substitutions of high conservation have a score of 0, substitutions of low conservation have a score of 5 and substitutions of intermediate conservation have a score of 1, 2, 3 or 4. Preferably substitution of the candidate residue is not an unconserved substitution, that is preferably (using the scoring system of FIG. 4) the candidate residue does not have conservation score (relative to cysteine) of 5. More preferably the candidate residue has a higher conservation relative to cysteine (e.g. a score of 4, 3, 2 and, more preferably, 1). The scoring system is described in the section entitled `Conservative Substitution` (below).

[0053] (h) Expression Level.

[0054] The thio-albumin may or may not be capable of being expressed at a level of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to the expression of an unmodified albumin (such as SEQ ID No. 1) from a suitable expression system, such as yeast (e.g. Saccharomyces, e.g. S. cerevisiae) or an Aspergillus. Relative expression levels can be determined, for example, by expression of the protein followed by quantification by SDS-PAGE, HPLC or Western Blotting.

[0055] (i) Conjugation Competence.

[0056] The thio-albumin may or may not have a high level of conjugation competence, for example at least 50, 60, 70, 80, 90, 95 or 100% relative to the conjugation competence of an albumin consisting of SEQ ID No. 1 having only one conjugation competent cysteine at Cys-34. Conjugation competence may be determined relative to any conjugatable molecule (conjugation partner) of interest, for example a bioactive molecule or a fluorescent dye. Determination may be through mass spectrometry analysis or quantification of the activity of the bioactive compound such as its fluorescence. An advantage of a thio-albumin having a high conjugation competence is that it may allow efficient conjugation of molecules to the thio-albumin. Conjugation competence may be measured with respect to time. Favoured thio-albumins may be (a) those which achieve maximal conjugation quickly or (b) slowly.

[0057] (j) Activity of Conjugated Compound.

[0058] The thio-albumin of the invention may be conjugated to a compound (conjugation partner), for example a bioactive compound, such that the compound has a high level of activity relative to its activity in an unconjugated state. Preferably, the conjugated compound shows at least 1, 10, 20, 40, 50, 60, 70, 80, 80 and most preferably 100% of its activity relative to its unconjugated state. An advantage of a conjugated compound with a high level of activity is that it reduces the quantity of conjugated compound required to achieve a desire effect, e.g. a desired therapeutic effect.

[0059] (k) Receptor Binding Capacity of Albumin

[0060] The conjugated- and/or non-conjugated thio-albumin may or may not have a receptor binding activity of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the receptor binding activity of human serum albumin (SEQ ID No. 1). Alternatively, the conjugated- and/or non-conjugated thio-albumin may or may not have a lower receptor binding activity for example at most 0, 10, 20, 30, 40, 50, 60, 70, 80 or 90% than human serum albumin. Receptor binding activity may be determined by assay, such as in relation to binding to FcRn.

[0061] FIG. 1. shows the scores of each amino acid residue of HSA (SEQ ID No. 1) for each of parameters (a) to (g). For clarity, in vivo, HSA is initially produced as a 609 amino acid protein in which the first twenty four amino acids are a leader sequence. The leader sequence is cleaved off to generate a 585 amino acid mature protein. Throughout this specification, the mature protein is referred to as SEQ ID No. 1. The structure of HSA model A106 disregards the first four residues and the last three residues of SEQ ID No. 1 because these are unresolved in the 3D model. Therefore, residue 1 of model A106 is equivalent to residue 5 of SEQ ID No. 1. Throughout this specification, all residues are cited with reference to SEQ ID No. 1, unless stated otherwise. The immature sequence of HSA (i.e. HSA with its natural C-terminal leader sequence) is provided in SEQ ID No. 102.

[0062] The column labels of FIG. 1. are detailed below:

[0063] Position in 1AO6: Refers to the amino acid position in the crystal structure of human serum albumin from the RCSD Protein Databank (PDB, http://www.rcsb.org/pdb/) with the entry with PDB identity 1AO6 or 1ao6 (Sugio, S., A. Kashima, et al. (1999). Protein Eng 12(6): 439-46). Note that compared to the mature HSA sequence (SEQ ID No1), the 1AO6 structure starts at residue 5S (with the first 4 amino acids absent from the structure) and finishes at 582A of SEQ ID No1 (with the last 3 amino acids absent from the structure). The amino acid positions used herein to describe positions to alter to generate conjugation competent cysteines are referring to the positions in SEQ ID No1, not 1ao6.

[0064] Position in Mature HSA: The amino acid position in SEQ ID No. 1 taken from the 585 residue secreted form of HSA, National Center for Biotechnology Information, ACCESSION: 1AO6_A VERSION GI:3212456 (24-SEP.-2008), Chain A, Crystal Structure Of Human Serum Albumin. (Sugio et al. (1999). Protein Eng 12(6): 439-46).

[0065] Position with Leader Sequence: Refers to the position in the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence.

[0066] Amino Acid: The standard one letter code for each of the 20 amino acids (e.g. A=Ala=Alanine).

[0067] % SASA: The solvent accessible surface area calculated for each residue, using the DSSP software described in Kabschand and Sander (1983). Biopolymers 22(12): 2577-637. Each solvent accessible surface area was divided by a standard value for the particular amino acid found in that position and multiplied by 100, thereby obtaining a percentage of the standard value for each residue. The standard solvent accessible surface areas for the 20 different amino acids are defined as (using one-letter codes for the amino acids): A=62, C=92, D=69, E=156, F=123, G=50, H=130, I=84, K=174, L=97, M=103, N=85, P=67, Q=127, R=211, S=64, T=80, V=81, W=126, Y=104).

[0068] B-Factor: The crystallographic B-factor value for the C-alpha atom was extracted directly from the PDB file. The B-factor is in column number 11 of the 1ao6 PDB file PDB, (http://www.rcsb.org/pdb/).

[0069] SS (Secondary Structure): The secondary structure determined for each residue using the DSSP software Kabsch and Sander (1983). Biopolymers 22(12): 2577-637. If the secondary structure is defined as H (Helix), B (isolated beta bridge) or E (Extended sheet), the residue is marked `1`, otherwise as `0`.

[0070] Align 1 (Mamm. W): The homology level for an alignment of various mammalian albumin family proteins with HSA (SEQ ID NO: 1), identified as P02768 compiled using MegAlign program (DNASTAR, Lasergene, version 8.0.2) based on Clustal W; six levels of homology are determined with the highest=100%, decreasing in 20% increments, to the lowest=0% (FIG. 2).

[0071] Adj. 100%'s (Align 1): The score according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with the mammalian albumins of FIG. 2. A score of 0 indicates the residue is not adjacent to a residue with 100% homology when HSA is aligned with the mammalian albumins of FIG. 2; a score of 1 indicates that the residue is adjacent to one residue with 100% homology when HSA is aligned with mammalian albumins; a score of 2 indicates that the residue is adjacent to two residue with 100% homology when HSA is aligned with mammalian albumins.

[0072] Align 2 (Var. Sps. V): The homology level for an alignment of various albumin family proteins with HSA (SEQ ID NO: 1), identified as P02768 compiled using MegAlign program (DNASTAR, Lasergene, version 8.0.2) based on Clustal V; six levels of homology are determined with the highest=100%, decreasing in 20% increments, to the lowest=0% (FIG. 3).

[0073] Polymorph: This identifies whether or not a polymorphism is known at the amino acid residue. Single amino acid polymorphisms of human serum albumin (SEQ ID NO: 1) were taken from Minchiotti et al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768, with amino acid positions taken from the unprocessed form of human serum albumin containing the 24 amino acid secretory leader sequence, and described using the standard one letter amino acid code (e.g. D25V refers to an aspartic acid being changed to a valine at position 1 in SEQ ID NO: 1).

[0074] Phenotype Change: A score representing the `severity` of phenotypic change derived from the sources of known phenotypic changes ('Polymorph.', referenced above) where; 0=no known phenotypic change, 1=a phenotypic change has been described for any of the mutations at this position compared to the residue in SEQ ID NO: 1, excluding a change resulting in decreased thermal stability, 2=a mutation at this position in SEQ ID NO: 1 is described `as causing reduced thermal stability.

[0075] Conserved Mutation vs. Cysteine: A score referring to how well conserved the amino acid is compared to cysteine, as derived from FIG. 4 (described herein), and ranging from 1 to 5 for mutations to cysteine. A score of 1 is assigned to the most conservative changes possible (e.g. alanine to cysteine), and ranging to a score of 5 for the lowest of conservation compared to a cysteine (e.g. histidine to cysteine).

[0076] Although the selection criteria can be used in any desired combination, four preferred groups of selection criteria (A, B, C, D) are described, by way of example only, below. Of these (A) and (B) may also be referred to as Selection Groups 1 and 2 (respectively):

[0077] (A) A particularly preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:

[0078] providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is `full length`, i.e. the mature amino acid sequence of the albumin);

[0079] selecting a candidate amino acid residue in the albumin sequence which corresponds to the first, second, third, fourth or fifth residue relative to the N- or C-terminus of the albumin sequence (of the model or of the amino acid sequence) or which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility (SASA) of at least 90%; B-factor score of at least 60; less than 80% homology to known mammalian albumins (e.g. FIG. 2); no adjacent residues with 100% homology to known mammalian albumins (e.g. FIG. 2); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above;

[0080] substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,

[0081] optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and

[0082] optionally preparing a polypeptide having the required amino acid sequence.

[0083] With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (A) include L585, D1, A2, D562, A364, A504, E505, T79 and E86 (in descending order of solvent accessibility) and are also shown in FIG. 5A.

[0084] (B) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:

[0085] providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is `full length`, i.e. the mature amino acid sequence of the albumin);

[0086] selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 90%; B-factor score of at least 40; less than 80% homology to known mammalian albumins (e.g. FIG. 2); no adjacent residues with 100% homology to known mammalian albumins (e.g. FIG. 2); no polymorphism with a known phenotypic change; and no unconserved amino acid change to cysteine with a score of 5 or above;

[0087] substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,

[0088] optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and

[0089] optionally preparing a polypeptide having the required amino acid sequence.

[0090] With reference to model 1A06 and SEQ ID No. 1, candidate residues identified by selection criteria (B) include D129, D549, A581, D121, E82, S270, Q397 and A578 (in descending order of solvent accessibility) and are also shown in FIG. 5B.

[0091] (C) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:

[0092] providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model, preferably the amino acid sequence is `full length`, i.e. the mature amino acid sequence of the albumin);

[0093] selecting a candidate amino acid residue in the albumin sequence (of the model or of the amino acid sequence) which (preferably in relation to the three dimensional model) fulfills the following conditions: not present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 50; less than 100% homology to known mammalian albumins (e.g. FIG. 2); less than 80% homology to the various albumins aligned in FIG. 3; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above;

[0094] substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue;

[0095] optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and

[0096] optionally preparing a polypeptide having the required amino acid sequence.

[0097] With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (C) are shown in FIG. 5C.

[0098] (D) Another preferred embodiment of the first aspect of the invention provides a method for designing and/or preparing a thio-albumin, the method comprising:

[0099] providing a three-dimensional model comprising at least one instance of an albumin sequence (preferably the three dimensional model relates to an amino acid sequence of an albumin and most preferably the the amino acid sequence of the albumin sequence is also provided, the amino acid sequence may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids which are not resolved at the C- and/or N-terminus of the three dimensional model);

[0100] selecting a candidate amino acid residue in the albumin sequence which (preferably in relation to the three dimensional model) fulfills the following conditions: present within secondary structure; surface accessibility of at least 80%; B-factor score of at least 30; less than 100% homology to known mammalian albumins (e.g. FIG. 2); less than 80% homology to the various albumins aligned in FIG. 3; no polymorphism known to cause thermal instability; and no unconserved amino acid change to cysteine with a score of 4 or above;

[0101] substituting one or more of the selected amino acid residues with cysteine or inserting cysteine at the N-side or C-side of the selected residue,

[0102] optionally making one or more additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, extension, addition or insertion; and

[0103] optionally preparing a polypeptide having the required amino acid sequence.

[0104] With reference to model 1AO6 and SEQ ID No. 1, candidate residues identified by selection criteria (D) are shown in FIG. 5D.

[0105] Since FIGS. 5A, 5B, 5C and 5D are selections from FIG. 1, the column headings are the same.

[0106] A candidate residue may be one or more of the cysteine residues involved in disulphide bonding present in the albumin molecule (in the case of HSA, SEQ ID No. 1, there are 17 disulphide bonds and therefore 34 cysteines involved in disulphide bonding). Two cysteines which are linked by a disulphide bond may be referred to as `counterparts`. In order to generate a conjugation competent cysteine, the candidate residue may be deleted or may be substituted with a different amino acid, particularly Ser, Thr, Val or Ala in order to create a free thiol at the partner cysteine. The 34 cysteine residues of SEQ ID No. 1 which are involved in disulphide bonding are C53, C62, C75, C91, C90, C101, C124, C169, C168, C177, C200, C246, C245, C253, C265, C279, C278, C289, C316, C361, C360, C369, C392, C438, C437, C448, C461, C477, C476, C487, C514, C559, C558 and C567. In relation to the invention, some of these 34 candidate residues are more favoured than others.

[0107] Cysteine residues were visually inspected using the PyMOL software (Warren L. DeLano "The PyMOL Molecular Graphics System." DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org), and the cysteines in the disulphide bonds were divided into 3 categories: [0108] those that can be replaced, for example, by serine, leaving its counterpart cysteine as a free thiol that has a high probability of being a conjugation site. These correspond to C75, C91, C124, C168, C169, C316, C360, C361, C567, C558. [0109] those that can be replaced by serine, leaving its counterpart as a free thiol that has a medium probability of being a conjugation site. These correspond to C90, C101, C177, C265, C279, C278, C289, C369, C392, C438, C476, C487, C514, C559. [0110] those that can be replaced by serine, leaving its counterpart as a free thiol that has a low probability of being a conjugation site. These correspond to C53, C62, C200, C246, C245, C253, C437, C448, C461, C477.

[0111] The judgment is based on surface accessibility and the orientation of the C-alpha -C-beta bond of the potential free thiol relative to the folded polypeptide. Using this judgment each of the cysteine residues of HSA were given a modification score and ranked as high, medium or low.

[0112] FIG. 6A, provides a list of all the cysteine residues which have a high modification score (right hand column), indicating that modification of a cysteine residue at this position would result in its counterpart cysteine providing a free thiol that has a high probability of being suitable for use as a conjugation site.

[0113] FIG. 6B, provides a list of the counterpart cysteines that that, when unpaired (thus providing a free thiol), have a high probability of being suitable for use as a conjugation site

[0114] The column labels for FIG. 6 are the same for those described for FIG. 1 with the addition of:

[0115] Modification Score: defined as `high`, `medium` or low' as described herein.

[0116] Disulphide Information: summarises disulphide pairing in SEQ ID No. 1.

[0117] (Polymorp.) Phenotype Change: summarises the columns labelled `Polymorph.` And `Phenotype Change` in FIG. 1.

[0118] Preferred cysteine residues for modification could be further selected based on the other information provided in FIG. 6A, such as assigned secondary structure, cysteine residues with no adjacent conserved residues (100% amongst mammalian albumins (aligned by Clustal W), no known polymorphisms causing phenotypic changes.

[0119] Alternatively cysteine residues for modification could be selected by examining the environment of the cysteine residue (containing a free thiol group) generated by modification of its counterpart cysteine residue provided in FIG. 6A, characteristics such as high % SASA may be preferred (FIG. 6B, fifth column).

[0120] For clarity, for albumins other than the human serum albumin of SEQ ID No. 1, equivalent residues are favoured for mutation. Such equivalent residues may or may not have identical residue numbers to those of SEQ ID No. 1 but would be clearly identifiable by the skilled person, for example with reference to homology alignments with SEQ ID No. 1 and other albumins such as those of FIG. 2 and/or FIG. 3. For example, in FIG. 2, the residue at positions at 160 of the horizontal `ruler` are equivalent but have differing residue numbers and, sometimes, are differing amino acids, e.g. L159 in human, W134 in goat fragment, L151 in Macacque and M159 in mouse. It is preferred that, for an alignment such as FIG. 2 or FIG. 3, equivalent residues are within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid of the candidate amino acid of SEQ ID No. 1. The `ruler` above the alignment indicates whether an amino acid is within 1 to 10 amino acids of a candidate amino acid of SEQ ID No. 1.

[0121] The selection criteria of Group (A) are more preferable than those of Group (B) which in turn are more preferable than those of Group (C) and in turn are more preferable than those of Group (D).

[0122] The method may or may not further comprise determining the receptor binding capacity and/or the conjugation competence of the polypeptide and optionally selecting a polypeptide which does or does not have a receptor binding capacity and/or conjugation competence.

[0123] `Preparing` a polypeptide may or may not include expressing the polypeptide in a host cell and/or purifying the polypeptide from the host or host cell media. The method may comprise favouring selection of residues meeting one or all of the following criteria: [0124] residues having high surface accessibility are preferred to those having low surface accessibility; [0125] conservative mutations from another amino acid to cysteine are preferred over non-conservative mutations;

[0126] Alternatively, or in addition, selection criteria as detailed throughout this specification may or may not be used to select residues in the method of the first aspect of the invention.

[0127] A second aspect of the invention provides a thio-albumin comprising a polypeptide sequence and/or polypeptide designed and/or produced according to the first aspect of the invention.

[0128] Preferably the polypeptide is a recombinant polypeptide. Preferably the polypeptide is an isolated and/or purified polypeptide. Preferably the polypeptide is synthetic and/or does not naturally occur in nature.

[0129] Specifically, the invention provides a polypeptide which has an amino acid sequence which is at least 60% identical to residues 1 to 585 of SEQ ID No. 1 or a fragment or fusion thereof, in which:

[0130] (a) at a position equivalent to position 34 of SEQ ID No. 1, there is a conjugation competent cysteine residue; and

[0131] (b) elsewhere in the polypeptide there is one or more conjugation competent cysteine residues, preferably 2 or more.

[0132] In addition the invention provides a conjugation competent polypeptide comprising an amino acid sequence which is at least 60% identical to residues 1 to 585 of SEQ ID No. 1, or a fragment or fusion thereof, in which:

[0133] (a) at a position equivalent to position 34 of SEQ ID No. 1, there is not a conjugation competent cysteine residue; and

[0134] (b) elsewhere in the polypeptide there is one or more conjugation competent cysteine residues, preferably 2 or more or 3 or more.

[0135] The polypeptide may or may not comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 conjugation competent cysteine residues.

[0136] More specifically, the polypeptide (which may be described in relation to a known albumin sequence such as SEQ ID No. 1) may or may not comprise one or more of:

[0137] (a) substitution of a non-cysteine amino acid with a cysteine at a position corresponding to a position equivalent to any of residues L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397 and A578 of SEQ ID No. 1;

[0138] (b) insertion of a cysteine at a position adjacent the N- or C-side of an amino acid which may or may not correspond to a position equivalent to any of residues L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397 and A578 of SEQ ID No. 1;

[0139] (c) a cysteine with a free thiol group at a position which may or may not correspond to any of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 which may or may not be generated by deletion or substitution of C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567.

[0140] (d) addition of a cysteine to the N-side of the N-terminal residue of an albumin sequence and/or to the C-side of the C-terminal residue of an albumin sequence such that the net result of the substitution, deletion, addition or insertion events of (a), (b), (c) and (d) is that the number of conjugation competent cysteine residues of the polypeptide sequence is increased relative to the polypeptide prior to the substitution, insertion, deletion and addition events. Within (a) to (d), above, the residues all of the residues are preferred. However, within each of (a), (b), (c) and (d), the residues are listed in order of decreasing preference.

[0141] A thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1), is modified to an amino acid which is not cysteine. For example, cysteine may or may not be replaced by an amino acid which has a relatively high conservation score (e.g. 1, 2 or 3 as calculated according to FIG. 4) such as alanine or serine. A thio-albumin may or may not include a polypeptide where one or more naturally occurring free-thiol group(s), such as cysteine-34 in HSA (SEQ ID No. 1) are present.

[0142] As detailed herein, the invention may be achieved by introducing cysteine residues by one or more of extension, addition, insertion, substitution or deletion.

[0143] An addition may be made by extension and/or insertion.

[0144] For example, one or more conjugation competent cysteines may or may not be created in an albumin by extension; e.g. by adding an extra cysteine residue to the N-terminus or C-terminus of the molecule, which may or may not be added as a single cysteine residue, or as a longer polypeptide which contains one or more conjugation competent cysteines. The cysteine residue(s) may be added immediately adjacent the N- or C-terminus of the albumin. Alternatively, there may be one or more other amino acid residues located between the N- and/or C-terminus of the albumin and the cysteine residue(s). When two or more cysteine residues are added, some or all of the added cysteines may be separated from each other by one or more other amino acids, for example by from 1 to 50 amino acids, such as from 1, 10, 20, 30, or 40 amino acids to from 10, 20, 30, 40, or 50 amino acids. A preferred N-terminal extension is the addition of Cys immediately adjacent the N-terminal of a mature albumin (i.e. albumin cleaved from its leader sequence). For example, for an albumin comprising or consisting of SEQ ID No. 1, Cys is preferably immediately N-terminal to the first Asp (D1). Such an albumin may be referred to as `Cys-albumin`, e.g. `Cys-HSA` (where HSA is Human Serum Albumin). Other preferred N-terminal extensions of albumins such as SEQ ID No. 1 include Cys-Ala-albumin such as Cys-Ala-HSA. A preferred C-terminal extension is the addition of Cys immediately adjacent the C-terminal of an albumin, such as a mature albumin. For example, for an albumin comprising or consisting of SEQ ID No. 1, Cys is preferably immediately C-terminal to the last Leu (L585) residue. Such an albumin may be referred to as `albumin-Cys`, e.g. HSA-Cys. Other preferred C-terminal extensions of albumins such as SEQ ID No. 1 include albumin-Ala-Cys, such as HSA-Ala-Cys. Polypeptides suitable for providing extensions, as described above, may be added or inserted to the C- or, N-side of the C- or N-terminal amino acid of the albumin, such as to the C-side of L585 in SEQ ID No. 1.

[0145] The polypeptide may or may not further comprise a further linker to which a conjugation partner, such as a bioactive compound, may be linked. For example a linker may comprise a primary amine such as a lysine.

[0146] One or more conjugation competent cysteines may or may not be created in an albumin by insertion; for example by adding one or more additional cysteines without removal of an amino acid residue from the albumin sequence, or by substituting one or more adjacent amino acids with a larger number of residues containing at least one cysteine, thus extending the overall length of the polypeptide. For example, a cysteine residue may be introduced immediately adjacent an albumin residue identified herein. The cysteine residue may be introduced as a single cysteine residue or within a polypeptide. The polypeptide may be from 2 to 50 amino acids long, preferably from 2, 10, 20, 30, or 40 to 10, 20, 30, 40 or 50 amino acids long.

[0147] Alternatively, or in addition, the invention includes substitution of one of the cysteine residues in one or more disulphides bond of an albumin with a different amino acid residue, so breaking the disulphide bond to leave an additional free thiol group. For example, a cysteine of one or more of the 17 naturally occurring disulphide bonds of HSA may be substituted to provide a conjugation-competent cysteine. Such a substitution causes the cysteine which has not been substituted to no longer have a disulphide binding partner and therefore provide a free thiol group. Conjugation competent cysteines may be provided from one or more of the naturally occurring disulphide bonds of an albumin such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the naturally occurring disulphide bonds of an albumin such as HSA (e.g. SEQ ID No. 1). For example, one cysteine residue which naturally forms a disulphide bond with another cysteine residue may or may not be substituted with a relatively conserved amino acid residue, particularly Ser, Thr, Val or Ala. With reference to SEQ ID No. 1, cysteine residues involved in disulphide bonding are C53, C62, C75, C91, C90, C101, C124, C169, C168, C177, C200, C246, C245, C253, C265, C279, C278, C289, C316, 0361, C360, C369, C392, C438, C437, C448, C461, C477, C476, C487, C514, C559, C558 and C567. Cysteine residues preferred for modification (i.e. deletion or substitution) may in particular correspond to C360, C316, C75, C168, C558, C361, C91, C124, C169 and/or C567 thus generating a conjugation competent cysteine at one or more of C369, C361, C91, C177, C567, C316, C75, C169, C124 and C558 of SEQ ID No. 1.

[0148] In addition, conjugation competent cysteines may or may not be created in albumin by deletion of one of the cysteines of a disulphide bond in the protein structure, so breaking the disulphide bond to provide an additional free thiol group.

[0149] Alternatively, or in addition, one or more of the cysteine residues present in the albumin molecule, but not involved in disulphide bonding (e.g. Cys-34 in the case of SEQ ID No. 1) may or may not be deleted (i.e. without substitution with a different amino acid) or may or may not be substituted with a different amino acid, particularly Ser, Thr, Val or Ala.

[0150] For a polypeptide comprising two or more conjugation competent cysteine residues, when the polypeptide is folded, the conjugation competent cysteine residues may or may not be relatively evenly distributed over the surface of the folded protein. The term `folded` includes folding of a polypeptide/protein into its natural configuration, for example the most thermodynamically stable folded configuration. An advantage of relatively even distribution is that it allows conjugation of two or more moieties to the thio-albumin without steric hindrance between two or more of the conjugated moieties. This has the advantage of minimising, and optionally eliminating, potential loss of activity due to issues such as steric hindrance between adjacent moieties (conjugation partners) which may be conjugated to the thio-albumin. Such moieties, for example bioactive molecules, may be relatively bulky.

[0151] Preferably the two or more conjugation competent cysteines are distributed over the surface of the thio-albumin molecule such that they are spaced as far from each other as possible, for example geometrically possible. Preferably the distance between two or more conjugation competent cysteines is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms. Preferably each conjugation competent cysteine is at least 10, 20, 30, 40, 50, 60, 70, or 80 Angstroms distant from all other conjugation competent cysteines in the molecule. The distance between two conjugation competent cysteines is preferably a distance which is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule, for example as shown in a model of an albumin. For example, the longest axis of SEQ ID No. 1 as shown in protein structure 1AO6 is approximately 85 Angstroms. Therefore, it is preferred that the two or more of the cysteine residues are at least 65, 70, 75 or most preferably 80 Angstroms apart. Most preferably each conjugation competent cysteine residue is at a distance of at least 80, 90, or 95% and most preferably 100% of the length of the longest axis of the folded albumin molecule.

[0152] Preferably the side chains of conjugation competent cysteines are directed away from each other and/or directed so that a moiety conjugated to the cysteine will be directed away from the centre of the albumin structure. This provides the advantage of preventing interactions between the conjugated moieties and/or the albumin moiety itself.

[0153] With reference to an amino acid sequence, candidate amino acid residues may be visually inspected using software such as PyMOL (Warren L. DeLano "The PyMOL Molecular Graphics System" DeLano Scientific LLC, San Carlos, Calif., USA. http://www.pymol.org). Candidate amino acids may be divided into categories based on their proximity to other members of that group. For example, candidate amino acids may be divided into 2, 3, 4, 5, 6, 7, 8, 9 or 10 categories. It is preferred that combinations of candidate amino acids are selected from different categories. That is, it is preferred that a thio-albumin contains one or fewer mutations from each category.

[0154] With reference to SEQ ID No. 1, PyMOL was used to analyse the candidate residues of FIGS. 5A and 5B in order to identify particularly favoured combinations of cysteine mutations. Such combinations may be used to design a thio-albumin having two or more conjugation competent cysteine residues. Selection Groups 1 and 2 correspond to the selection criteria (A) and (B) (respectively) from FIGS. 5A and 5B of the selection method described herein. Selection Group 3 corresponds to the residues identified in FIG. 6B. Particularly favoured residues are given in FIG. 6A and 6B in which the column headings are the same as those in FIG. 1 with the addition of `Selection Group` and `Proximity Group` as described herein.

[0155] The results of the analysis are given in FIG. 10 in which column headings are the same as those used in FIG. 1, and FIG. 6 with the addition of:

[0156] Proximity Group: allocation of a proximity group as described herein to describe subsets of sites within HSA (specifically SEQ ID No. 1).

[0157] For example, candidate amino acid residues selected in Selection Group 1 (listed in FIG. 5A) were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of Selection Group 1. Five groups were generated (labeled A to E in FIG. 10 `proximity group`, right hand column), four were generated by visual inspection. Group E contains amino acid residues not visible in 1AO6 which are known to be in the N-terminal region. In addition it was observed that cys-34 is present in the proximity group A.

[0158] Similarly amino acid residues selected in Selection Group 2 (listed in FIG. 5B) were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of Selection Group 2. Five groups were generated (labeled F-J in FIG. 10 `proximity group`, right hand column)

[0159] Similarly, the preferred free cysteine residues selected in Selection Group 3 (listed in FIG. 6B), which can be generated by mutations causing disruption of disulphide bonds were visually inspected using the PyMOL software, and the amino acids selected were divided into categories based on their proximity to other members of that group. Four groups were generated (labeled K-N in FIG. 10 `proximity group`, right hand column). When referring to the residues of selection group 3, the cited residues are the resultant conjugation competent cysteines (e.g. FIG. 6B). In order to generate such a conjugation competent cysteine it is clear that the counterpart cysteine (e.g. FIG. 6A) in the disulphide bond should be removed for example by deletion or substitution.

[0160] When a combination of two or more mutations is desired, amino acid residues which occur in different `proximity` groups (e.g. with reference to SEQ ID No.1) may be preferred over those that occur within the same proximity group. For SEQ ID No. 1, there are 14 proximity groups (i.e. A to N). It is preferred that, for a thio-albumin having two or more conjugation competent cysteines, there is zero or one conjugation competent cysteine defined from each of the 14 groups. That is, it is preferred that such a thio-albumin does not contain two or more conjugation competent cysteines falling within the same group. A large number of permutations exist which meet this criterion.

[0161] For example, for a thio-albumin variant containing two free thiol groups residues based on selection criteria that generated Selection Group 1, then T79+A364, in which one residue is selected from proximity group A to combine with A364 in proximity group C, would be preferred over T79+E86 which both occur in proximity group A.

[0162] For combinations including cysteine-34, it is preferred not to select residues from proximity groups A, F or K. That is, it is preferred to select residues from one or more of proximity groups B to E, G to J and L to N.

[0163] Examples of preferred mutations selected from within Selection Group 1 may include the following: [0164] For 2 amino acid residues selected from Selection Group 1; amino acid residues from proximity groups A+C are preferred, such as T79+A364, Similarly, amino acid residues selected from proximity groups C+E, such as A364+D1 are also preferred. Also, amino acid residues from proximity groups D+E, such as L585+A2 or + the C-side of L585+A2, or from G+I, such as S270+A581, are preferred. [0165] For 3 amino acid residues selected from Selection Group 1; amino acid residues which occur in proximity groups A+C+B are preferred such as T79+A364+D562. Similarly, amino acid residues selected from proximity groups B+C+E, such as D562+A364+A2 or D562+A364+D1, are also preferred [0166] For 4 amino acid residues selected from Selection Group 1; amino acid residues which occur in proximity groups A+C+B+D such as such as T79+A364+D562+A504, or alternatively T79+D562+A364+L585 are preferred. Even more preferred are amino acid residues selected from proximity groups A+B+C+E, such as C34+D562+A364+A2 T79+D562+A364+D1. [0167] For 5 amino acid residues selected from Selection Group 1; amino acid residues which occur in proximity groups A+C+B+D+E such as T79+D562+A364+L585+D1 are preferred. Similarly, E86+D562+A364+A504+A2 are also preferred. [0168] The above mentioned albumin variants may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position in another albumin.

[0169] Examples of preferred mutations selected from within Selection Group 2 may include the following: [0170] For 2 amino acid residues selected from Selection Group 2; amino acid residues which occur in proximity groups G+I such as S270+A581 are preferred. Alternatively, amino acid residues which occur in proximity groups G+H such as S270+D129 are preferred. [0171] For 3 amino acid residues selected from Selection Group 2; amino acid residues which occur in proximity groups G+I+F such as S270+A581+E82 are preferred. Alternatively, amino acid residues which occur in proximity groups G+I+H such as S270+A581+D129 are preferred. [0172] For 4 amino acid residues selected from Selection Group 2; amino acid residues which occur in proximity groups G+I+F+H such as S270+A581+E82+D129. [0173] For 5 amino acid residues selected from Selection Group 2; amino acid residues which occur in proximity groups G+I+F+H+J such as S270+A581+E82+D129+Q397 are preferred. However, D549 is not preferred in combination with mutations A578, A581. Also, mutations to D549, A578, A581 or are not preferred in combination with mutation of L585 from Selection Group 1.

[0174] Examples of preferred site selected from within Selection Group 3 for the conjugation competent free-thiols may include the following: [0175] For 2 amino acid residues selected from Selection Group 3; amino acid residues which occur in proximity groups M+L are preferred, such as C369+C177. Similarly, C361+C124 are also preferred. [0176] More than two mutations disrupting disulphide bonds are less preferred. [0177] The above mentioned albumin variants may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position in another albumin.

[0178] Combinations of sites from Selection Groups 1, 2 and 3 can also be made, where sites from Selection Group 1 are typically preferred to sites from Selection Group 2, which are typically preferred to sites selected from Selection Group 3.

[0179] Examples of sites from Selection Groups 1+2 may include residues from proximity groups C+I, such as A364+A581. Alternatively, residues from proximity groups A+G+I, such as C34+S270+A581, from proximity groups A+H+G+I, such as C34+D129.degree.S270+A581, from proximity groups A+C+I, such as T79+A364+A581, or residues from proximity groups C+I+H such as A364+A581+D129 are also preferred.

[0180] Examples of sites from Selection Groups 1+3 may include residues from proximity groups A+L+M, such as C34+C169+C316, from proximity groups C+L, such as A364+C177 are preferred. Alternatively, residues from proximity groups B+M, such as D562+C369 are preferred.

[0181] Examples of sites from Selection Groups 2+3 may include residues from proximity groups H+M, such as D129+C369 are preferred. Alternatively, residues from proximity groups I+M, such as A581+C369 are preferred.

[0182] Examples of sites from Selection Groups 1+2+3 may include residues from proximity groups A+H+M+D, such as C34+D129+C360+L585, from proximity groups B+H+M, such as D562+D129+C369 are preferred.

[0183] The above combinations are generally more preferred than combinations of residues from the following sets of proximity groups: (i) A, K and F; (ii) B, D, I, J and N; (iii); C and M; (iv) H and L.

[0184] The above albumin variants of the invention may or may not further comprise a cysteine at Cys34 of SEQ ID No. 1, or at an equivalent position, if based on an albumin other than SEQ ID No. 1.

[0185] A skilled person will appreciate that the sites for introduction of more than one free thiol group (conjugation competent cysteine) have been selected from Selection Groups 1, 2 and 3. However, this approach may also be used to select sites for the introduction of more than one free thiol group from other residues selected from SEQ ID No1, i.e. he could use the disclosed method to generate other useful selection groups.

[0186] A preferred thio-albumin comprises SEQ ID No. 1 with Cys at positions 2 and 585 in addition to the naturally occurring Cys at position 34 (SEQ ID No. 78, construct `TA33`). A more preferred thio-albumin comprises SEQ ID No. 1 with Cys at positions 2, 364, 562, 585 in addition to the naturally occurring Cys at position 34 (SEQ ID No. 82, construct `TA38`). Thio-albumins comprising three or four of the Cys at positions 2, 364, 562 and 585 may also be preferred.

[0187] The polypeptide may or may not comprise at least one mutation that reduces glycosylation.

[0188] A third aspect of the invention provides a polynucleotide which encodes the polypeptide according to the invention. The polynucleotide may or may not be codon-optimised relative to the host from which it is to be expressed. SEQ ID No. 2 provides the usual coding sequence of HSA (SEQ ID No. 1). SEQ ID No. 3 provides a coding sequence of HSA (SEQ ID No. 1) which is codon-optimised for expression from S. cerevisiae. SEQ ID No. 2 or 3 may be mutated in order to provide a polynucleotide which encodes a polypeptide according to the invention. Preferably the polynucleotide is synthetic and/or recombinant. Preferably the polynucleotide is an isolated polynucleotide. The polynucleotide may encode an HSA with or without a leader sequence. For example, the polynucleotide may encode an HSA with the natural leader sequence of HSA (amino acids 1 to 24 of SEQ ID No. 102) or an HSA with a fusion leader sequence (amino acids 1 to 24 of SEQ ID No. 49).

[0189] A fourth aspect of the invention provides a plasmid comprising the polynucleotide of the third aspect of the invention. The plasmid may be a 2 micron based plasmid such as those described in WO2005/061719, WO2005/061718 and WO2006/067511 (all incorporated herein by reference). The plasmid may exhibit enhanced chaperone activity, for example through over expression of a chaperone, particularly PDI.

[0190] A fifth aspect of the invention provides an expression system such as a host cell comprising a polynucleotide according to the third aspect of the invention and/or a plasmid of the fourth aspect of the invention. Preferably the host cell is a mammalian cell such as a human or bovine cell, or a fungal cell such as a yeast cell. Alternatively, the host cell may be a bacterial cell such as a Bacillus or Escherichia coli or a viral cell such as Baculovirus or a plant cell such as a rice e.g. Oryza sativa. Most preferably, the cell is a yeast cell such as a Saccharomyces (e.g. S. cerevisiae), a Pichia or an Aspergillus cell.

[0191] A sixth aspect of the invention provides a conjugate which comprises a conjugation partner, such as a bioactive compound, and a polypeptide according to the invention, wherein the conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide. The conjugation partner may be a therapeutic, diagnostic or imaging compound such as those mentioned herein. The conjugate may comprise 2 or more, for example 2, 3, 4, 5, 6, 7,8, 9 or 10, conjugation partners which may each be different and/or may be multiple copies of the same compound. Preferably, each conjugation partner is linked to the polypeptide through a conjugation competent cysteine residue of the polypeptide, however conjugation partners may be linked by other means for example by a genetic fusion or covalent bonds to non-cysteine amino acids such as lysine.

[0192] A seventh aspect of the invention provides a method of producing a polypeptide of the invention comprising:

[0193] (a) culturing a host cell according to the invention under conditions that allow expression of the polypeptide; and

[0194] (b) recovering the polypeptide from the host cell and/or from host cell growth medium.

[0195] Accordingly, the present invention also provides a method for producing a polypeptide (or protein) of the invention, the method comprising: (a) providing a host cell of the invention comprising a polynucleotide encoding protein product of choice as defined above; and (b) growing the host cell (for example, culturing the host cell in a culture medium); thereby to produce a cell culture or recombinant organism comprising an increased level of the protein product of choice compared to the level of production of the protein product of choice achieved by growing (for example, culturing), under the same conditions, the same host cell that has not been genetically modified to cause over-expression of one or more helper proteins.

[0196] The step of growing the host cell may or may not involve allowing a host cell derived from a multicellular organism to be regrown into a multicellular recombinant organism (such as a plant or animal) and, optionally, producing one or more generations of progeny therefrom.

[0197] The method may or may not further comprise the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.

[0198] The production method may comprise linking a conjugation partner to the polypeptide of the invention through a conjugation competent cysteine residue of the polypeptide. Suitable conjugation methods and conjugation partners are described herein.

[0199] An eighth aspect of the invention provides a composition comprising a conjugate according to the invention and at least one pharmaceutically acceptable carrier and/or diluent.

[0200] A ninth aspect of the invention provides a method for making a pharmaceutical ingredient and/or a pharmaceutical product comprising making a thio-albumin according to the present invention, optionally conjugating a further molecule to the thio-albumin, optionally formulating the resultant conjugate with a pharmaceutically acceptable diluent and/or carrier and optionally preparing the product in unit dosage form.

[0201] A tenth aspect of the invention provides use of a polypeptide according to the invention for the production of a thio-albumin-conjugate.

[0202] An eleventh aspect of the invention provides use of a conjugate according to the invention and/or produced by a method according to the invention for treatment of disease, treatment of illness and/or diagnosis.

[0203] A twelfth aspect of the invention provides a gel comprising one or more albumins according to the invention. The gel may be formed by any suitable method. For example the gel may be formed by incubating an albumin solution, or suspension, at a suitable temperature e.g. room temperature (15 to 25.degree. C., such as 20.degree. C.) or body temperature (36 to 38.degree. C., preferably 36.9.degree. C.). A gel may be used to coat medical devices, such as a stent. A gel may be used in or on a wound dressing. The albumin may be applied to the medical device or wound dressing before or after it has gelled. The albumin may be applied ex situ or in situ (e.g. applied to a medical device or dressing before, after or during its application on to or insertion into a human or animal body).

[0204] The polypeptides and/or conjugates of the invention may be used to prepare nanoparticles which may be used, for example, in angiogenic applications, anti-angiogenic applications and to coat a medical device such as a stent. Nanoparticles are effective at targeting, for example to non tight-junctions, and therefore can be useful for targeting tumours such as cancerous tumours. Nanoparticles can also be useful to target antigen in order to provoke an immune response since nanoparticles are particularly susceptible to engulfment and presentation by phagocytes. The invention provides nanoparticles consisting only of thio-albumin according to the invention which may or may not be conjugated to a moiety (conjugation partner). The invention also provides nanoparticles comprising thio-albumin according to the invention, which may or may not be conjugated to a moiety, and one or more other constituents of a nanoparticle which may or may not be albumin related. In a preferred embodiment, a thio-albumin according to the invention comprises at least two conjugation competent cysteine residues located on the surface of the polypeptide. Such a thio-albumin may be used for the preparation of nanoparticles in which one or more conjugation competent cysteine residues may be used in the formation of a nanoparticle and one or more conjugation competent residue is used for conjugation to a conjugation partner, for example to a bioactive molecule.

[0205] The invention relates to all albumins. Whilst preferred residues have been identified in relation to SEQ ID No. 1, the skilled person would be able to identify equivalent residues in other albumin sequences, such as the albumins disclosed in FIGS. 2 and 3, and understand that mutations of albumins (other than SEQ ID No. 1) at such equivalent residues are part of the invention. Equivalent residues can be identified by, for example, homology alignment with SEQ ID No. 1. A residue in an albumin other than SEQ ID No. 1 may or may not have an identical residue coordinate to its equivalent residue in SEQ ID No. 1. Thus the invention provides thio-albumins based on any albumin sequence, such as the sequences shown in Table 1 and, more preferably, those shown in FIG. 2 and/or 3. `Based on` includes modification of an albumin sequence to introduce one or more additional free-thiols.

[0206] Recombinant albumins can offer advantages over animal-derived albumins by having a higher level of conjugation-competent free thiol groups,' and can be manufactured without the risk of contamination with pathogenic prions and viruses. An advantage of a thio-albumin conjugate is that the thio-albumin part may be prepared separately to a conjugation partner. Therefore, one batch of thio-albumin may be used to produce many different thio-albumin conjugates. Also, the individual components of the conjugate can be manufactured by different methods and therefore are not restricted to a single method, such as heterologous protein expression in a host cell such as a yeast. Furthermore, a thio-albumin may comprise multiple conjugation sites and therefore a single thio-albumin may be conjugated to more than one type of conjugation partner (e.g. therapeutic agent, diagnostic agent, targeting agent, imaging agent) and/or to multiple copies of one or more types of conjugation partner. The ability to conjugate the thio-albumin to different types of conjugation partners allows the provision of a multi-functional species. The ability to conjugate the thio-albumin to multiple copies of a conjugation partner allows the concentration of molecule to be increased and therefore increase the amount, or volume, of thio-albumin conjugate required for a given purpose relative to a conjugate having only a single copy of the conjugation partner. Advantages of delivering drugs via an albumin fusion protein are discussed in Osborn, et al. (2002). J Pharmacol Exp Ther 303(2): 540-8. It is expected that delivery of drugs via a conjugation of the invention would have similar advantages.

[0207] Further details which may or may not be used in accordance with the invention are described below:

Three Dimensional (3D) Models

[0208] The above disclosure has been made in relation to the albumin model known as 1AO6 (Protein Data Bank) which relates to SEQ ID No. 1. FIG. 1 gives the amino acid residues for 1AO6.

[0209] However, the invention relates to all albumins and their structures. Structures of albumin are available to the skilled person, for example the atomic coordinates for the tertiary structure of human albumin are available at the GenBank DNA database at www.ncbi.nlm.nih.gov.

[0210] Structures may be viewed using suitable software such as RasM.1 Chime (Sayle, TIBS 20, 374, 1995). Available albumin coordinates include: [0211] 1AO6, 1BMO (Sugio et al. (1999). Protein Enq 12(6): 439-46), which was among the top 17 requested proteins. [0212] 1 UOR, He & Carter (1992). Nature 358(6383): 209-15. [0213] 1bj5 and 1bke, Curry et al. (1998). Nat Struct Biol 5(9): 827-35. [0214] 1e7a,1e7b, 1e7c, Bhattacharya et al. (2000). J Biol Chem 275(49): 38731-8. [0215] 1e7e, 1e7f, 1e7g, 1e7h and 1e7i, Bhattacharya et al. (2000). J Mol Biol 303(5): 721-32. [0216] 1GNJ, Petitpas et al. (2001). J Mol Biol 314(5): 955-60. [0217] 1HA2 and 1H9Z Petitpas et al. (2001). J Biol Chem 276(25): 22804-9.

Albumin

[0218] The albumin used in the invention may be a naturally occurring albumin, an albumin-related protein or a variant thereof such as a natural or engineered variant. Variants include polymorphisms, fragments such as domains and sub-domains, fragments and/or fusion proteins. An albumin, of this invention, may comprise the sequence of an albumin protein obtained from any source. Typically the source is mammalian such as human or bovine. In one preferred embodiment the serum albumin is human serum albumin ("HSA"). The term "human serum albumin" includes a serum albumin having an amino acid sequence naturally occurring in humans, and variants thereof. Preferably the albumin has the amino acid sequence of SEQ ID No. 1 or a variant or fragment thereof, preferably a functional variant or fragment thereof. The HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 0 073 646 and EP 0 286 424. A fragment or variant may or may not be functional. For example, a fragment or variant may retain the ability to bind to an albumin receptor such as FcRn to at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the ability of the parent albumin (from which the fragment or variant derives) to bind to the receptor. Relative binding ability may be determined by methods known in the art such as surface plasmon resonance studies.

[0219] The albumin may be a naturally-occurring polymorphic variant of human albumin or of a human albumin analogue. Generally, variants or fragments of human albumin will have at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, (preferably at least 80%, 90%, 95%, 100%, 105% or more) of human albumin's ligand binding activity (for example FcRN-binding), mole for mole.

[0220] The "albumin" may comprise the sequence of bovine serum albumin. The term "bovine serum albumin" includes a serum albumin having an amino acid sequence naturally occurring in cows, for example as taken from Swissprot accession number P02769, and variants thereof as defined herein. The term "bovine serum albumin" also includes fragments of full-length bovine serum albumin or variants thereof, as defined herein.

[0221] A number of proteins are known to exist within the albumin family; a non-exclusive list is shown in Table 1, below. The list indicates full-length of sequences including the mature protein and leader sequence (unless indicated otherwise).

[0222] The albumin may comprise the sequence of an albumin derived from one of serum albumin from dog (e.g. see Swissprot accession number P49822-1), pig (e.g. see Swissprot accession number P08835-1), goat (e.g. as available from Sigma as product no. A2514 or A4164),), cat (e.g. see Swissprot accession number P49064-1), chicken (e.g. see Swissprot accession number P19121-1), ovalbumin (e.g. chicken ovalbumin) (e.g. see Swissprot accession number P01012-1), turkey ovalbumin (e.g. see Swissprot accession number O73860-1), donkey (e.g. see Swissprot accession number Q5XLE4-1), guinea pig(e.g. see Swissprot accession number Q6WDN9-1), hamster (see DeMarco et al. (2007). International Journal for Parasitology 37(11): 1201-1208), horse (e.g. see Swissprot accession number P35747-1), rhesus monkey (e.g. see Swissprot accession number Q28522-1), mouse (e.g. see Swissprot accession number P07724-1), pigeon (e.g. as defined by Khan et al, 2002, Int. J. Biol. Macromol., 30(3-4),171-8), rabbit (e.g. see Swissprot accession number P49065-1), rat (e.g. see Swissprot accession number P02770-1) and sheep (e.g. see Swissprot accession number P14639-1) and includes variants and fragments thereof as defined herein.

[0223] The albumin may comprise the sequence of an albumin such as a serum albumin or an ovalbumin, for example those shown in Table 1, below, and includes variants and fragments thereof as defined herein.

TABLE-US-00001 TABLE 1 Albumins from various species Identity to SwissProt SEQ ID Accession NO: 1 Length Protein Common Name Species No (Clustal V) (aa) SA African clawed Xenopus laevis P08759-1 37.3 608 frog SA Bovine Bos taurus (SEQ P02769-1 76.1 607 ID No. 94) SA Cat Felis catus (SEQ P49064-1 82.2 608 ID No. 95) SA Chicken Gallus gallus P19121-1 46.5 615 (Version 2 text) SA Cobra ALB Naja kaouthia Q91134-1 30.9 614 SA Dog Canis lupus P49822-1 80.3 608 familiaris (SEQ ID No. 96) SA Donkey Equus asinus Q5XLE4-1 76.6 607 (SEQ ID No. 97) SA European water Rana shqiperica Q9YGH6-1 31.5 603 frog SA Blood fluke Schistosoma AAL08579 76.0 608 mansoni Q95VB7-1 SA Mongolian Gerbil Meriones O35090-1/ 73.2 609 unguiculatus (SEQ JC5838 ID No. 98) SA Goat Capra hircus (SEQ B3VHM9-1 74.3 607 ID No. 99) SA Guinea Pig Cavia porcellus Q6WDN9-1 73.0 608 (SEQ ID No. 100) SA Horse Equus caballus P35747-1 76.4 607 (SEQ ID No. 101) SA Human Homo sapiens P02768-1 100.0 609 (SEQ ID No. 102) SA Australian Neoceratodus P83517-1 22.8 101 Lungfish fosteri (NL) SA Macaque Macaca mulatta Q28522-1 93.5 608 (Rhesus (SEQ ID No. 103) Monkey) SA Mouse Mus musculus P07724-1 72.4 608 (SEQ ID No. 104) Version 3. SA North American Rana catesbeiana P21847-1 36.1 382 bullfrogs (NL) SA Pig Sus scrofa (SEQ P08835-1 75.6 607 ID No. 105) Version 2 SA Rabbit Oryctolagus P49065-1 75.3 608 cuniculus (SEQ ID Version 2 No. 106) SA Rat Rattus norvegicus P02770-1 73.4 608 (SEQ ID No. 107) Version 2. SA Salamander Ambystoma Q8UW05-1 38.8 626 maculatum SA Salmon ALB1 Salmo salar P21848-1 25.0 608 SA-2 Salmon ALB2 Salmo salar Q03156-1 24.8 608 SA Sea lamprey Petromyzon Q91274-1 16.9 1423 marinus SA Sea lamprey Petromyzon O42279-1 17.4 551 marinus -AS SA Sheep Ovis aries (SEQ ID P14639-1 75.3 607 No. 108) SA Sumatran Pongo abelii Q5NVH5-1 100.0 609 Orangutan SA Tuatara Sphenodon Q8JIA9-1 43.8 527 punctatus (NL) SA Western clawed Xenopus (Silurana) Q6DJ95-1 10.5 572 frog tropicalis (NL) OA Chicken Gallus gallus P01012-1 10.9 383 Version 2 (NL) OA Turkey Meleagris O73860-1 11.4 386 gallopavo Version 3. (NL) SA: Serum albumin, SA-2: Serum albumin-2, OA: Ovalbumin, NL: No Leader sequence

[0224] Many naturally occurring mutant forms of albumin are known. Many are described in Peters, (1996, All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, Inc., San Diego, Calif., p.170-181). A variant as defined herein may be one of these naturally occurring mutants such as those described in Minchiotti at al. (2008). Hum Mutat 29(8): 1007-16., and http://www.uniprot.org/uniprot/P02768,.

[0225] A "variant albumin" refers to an albumin protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin or a fatty acid such as a long-chain fatty acids, for exampleoleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and/or palmitoleic (C16:1)), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein, e.g. the protein from which the variant is derived. Such characteristics may be used as additional selection criteria in the invention.

[0226] Typically an albumin variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with a naturally occurring albumin such as SEQ ID No. 1. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program or the Clustal V program and therefore allow calculation of % homology between sequences of a multiple alignment and/or calculation of % identity between sequences of a pairwise alignment. The parameters used may be as follows:

[0227] Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM Custal W: Pairwise alignment parameters: `Slow-Accurate`, Gap Penalty: 10, Gap Length: 0.1, Protein Weight Matrix: Gonnet 250, DNA Weight Matrix: IUB. Multiple Alignment Parameters: Gap penalty 10.00, gap length penalty 0.20, Delay Divergent Seqs(%) 30, DNA transition weight 0.50, Protein weight matrix=Gonnet series, DNA weight matrix=IUB.

[0228] Clustal V: Pairwise alignment parameters: Ktuple: 1, Gap Penalty: 3, Window: 5, Diaganols: 5; Multiple alignment parameters: Gap penalty 10, gap length penalty 10.

Conservative Substitution

[0229] As used herein, the term "conservative" amino acid substitutions refers to substitutions made within the same group, and which typically do not substantially affect protein function. By "conservative substitutions" is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made by techniques well known in the art, such as by site-directed mutagenesis as disclosed in U.S.4 Pat. No 4,302,386 issued 24 Nov. 1981 to Stevens, incorporated herein by reference.

[0230] In one embodiment, the Venn diagram of FIG. 4 may be used to determine conservative amino acid substitutions: Using FIG. 4., a conservation mutation score (ranging from 0 to 5) may be calculated. A score of 0 is the highest conservation, which, for cysteine, is only assigned for substitution of a cysteine residue with another cysteine residue. For changes from any other amino acid to a cysteine, the score may be 1, 2, 3, 4, 5. A score of 1 is a more conservative substitution that a score of 2, 3, 4 or 5. A score of 5 is assigned to the lowest conservation between a substituted amino acid and the cysteine. The score of 0 to 5 is calculated from FIG. 4 as the number of boundaries (i.e. lines) crossed to go from cysteine to the appropriate amino acid. Thus the score for cysteine is 0 as no boundaries are crossed. Likewise, the score of aspartic acid (D) is 3, since 3 boundaries are crossed.

[0231] The conservation mutation score (with respect to FIG. 4) for the 20 different amino acids are defined as (using one-letter codes for the amino acids): A=1, C=0, D=3, E=4, F=4, G=2, H=5, I=4, K=4, L=4, M=3, N=2, P=3, Q=3, R=5, S=1, T=1, V=3, W=3, Y=3. With reference to FIGS. 1, 5A, 5B, 5C, and 5D, these scores are provided for each of the amino acid residues in the column labelled `Conserved Mutation to Cysteine`. Using the conservation mutation score residues with a score of 3 or less, i.e. aspartic acid methionine, proline, glutamine, valine, tryptophan, tyrosine, glycine, asparagine, alanine, serine and threonine are preferred since they are relatively conserved with cysteine. More preferred are those amino acids with a score of 2 or less i.e. glycine, asparagine, alanine, serine, threonine. Most preferred are those with a score of 1, i.e. alanine, serine, threonine. Similarly using the conservation mutation score system of FIG. 4, residues with a score of 4 or more, i.e. glutamic acid, phenylalanine, isoleucine, lysine, leucine, histidine and arginine are less preferred and may not be preferred at all.

[0232] Alternatively, or in addition, "conservative" amino acid substitutions refers to substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).

[0233] For example, a conservative substitution of alanine-2 in SEQ ID No 1 can include glycine or serine. Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group. For example, a non-conservative substitution could include the substitution of a polar amino acid for a hydrophobic amino acid.

Fragment

[0234] The term "fragment" as used herein includes any fragment of full-length albumin or a variant thereof, so long as at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein. A fragment will typically be at least 50 amino acids long. A fragment may comprise at least one whole sub-domain of albumin. Domains of HSA have been expressed as recombinant proteins (Dockal et al., 1999, J. Biol. Chem., 274, 29303-29310), where domain I was defined as consisting of amino acids 1-197, domain II was defined as consisting of amino acids 189-385 and domain III was defined as consisting of amino acids 381-585. Partial overlap of the domains occurs because of the extended a-helix structure (h10-h1) which exists between domains I and II, and between domains II and III (Peters, 1996, op. cit., Table 2-4). HSA also comprises six sub-domains (sub-domains IA, IB, IIA, IIB, IIIA and IIIB). Sub-domain IA comprises amino acids 6-105, sub-domain IB comprises amino acids 120-177, sub-domain IIA comprises amino acids 200-291, sub-domain IIB comprises amino acids 316-369, sub-domain IIIA comprises amino acids 392-491 and sub-domain IIIB comprises amino acids 512-583. A fragment may comprise a whole or part of one or more domains or sub-domains as defined above, or any combination of those domains and/or sub-domains. A fragment may comprise or consist of at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of an albumin or of a domain of an albumin.

[0235] Additionally, single or multiple heterologous fusions comprising any of the above; or single or multiple heterologous fusions to albumin, or a variant or fragment of any of these may be used. Such fusions include albumin N-terminal fusions, albumin C-terminal fusions and co-N-terminal and C-terminal albumin fusions as exemplified by WO 01/79271.

Homology

[0236] FIGS. 2 and 3 show alignments of various albumin family proteins with HSA (SEQ ID NO: 1), identified as `P02768`. The protein sequences include the albumin leader sequence. These alignments can be used to identify conserved regions and amino acid residues corresponding to those in HSA selected as described above. One or both alignments can also be used to assign a homology score to an amino acid residue in an albumin sequence.

[0237] An example of such a procedure is the MegAlign program (version 8.0.2) developed by DNASTAR Inc., part of the Lasergene suite, based on Hein, J. J. (1990) "Unified approach to alignment and phylogenies." In Methods in Enzymology, Vol. 183: pp. 626-645. Using the Jotun Hein Method and the settings GAP PENALTY=11, GAP LENGTH PENALTY=3 for multiple alignments and KTUPLE=2 for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal V Method and the settings GAP PENALTY=10, GAP LENGTH 10 for multiple alignments and KTUPLE=1. GAP PENALTY=3, WINDOW=5 and DIAGONAL=5 for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal W Method and the settings GAP PENALTY=10, GAP LENGTH PENALTY=0.2, DELAY DIVERGENCE=30 DNA transition=0.5 and using GONNET SERIES for Protein Weight matrix and IUB for DNA Weight matrix for multiple alignments, and Slow accurate, GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and using GONNET SERIES for Protein Weight matrix and IUB for DNA Weight matrix for pairwise alignments a series of percentage identity values can be calculated. Alternatively the Clustal V method may be used (above).The alignment of two amino acid sequences may also be determined by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman and Wunsch (1970) "A general method applicable to the search for similarities in the amino acid sequence of two proteins." J. Mol. Biol. 48, 443-453. The substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.

[0238] FIG. 2 shows an alignment of sixteen mammalian albumin family proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal W. The protein sequences include the albumin leader sequence. Each sequence is labelled with the animal from which it derives and its database accession number.

[0239] FIG. 3 shows alignments of thirty three albumin family (both mammalian and non-mammalian) proteins including HSA (SEQ ID NO: 1, identified in the alignment as P02768) compiled using MegAlign program (version 8.0.2) based on Clustal V. The protein sequences include the albumin leader sequence.

[0240] Homology may be determined with reference to FIG. 2 and/or FIG. 3. The degree of identity between a given amino acid sequence and SEQ ID NO: 1 may be calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the shorter of the two sequences. The result may be expressed in percent identity. An exact match occurs when the two sequences have identical amino acid residues in the same positions of the overlap. The length of a sequence is the number of amino acid residues in the sequence.

Conservation of Adjacent Residues

[0241] In addition, individual amino acid residues of HSA can be ranked according to their conservation to the amino acids of other albumin family proteins at the same position. FIG. 1, column labelled `Align 1 (Mamm. W ('mammalian, Clustal W)) provides the homology level for each position of SEQ ID No. 1 as calculated by the alignment of mammalian albumins given in FIG. 2. The homology level score may be calculated. One method to score the homology level, which is used herein, is calculated by using the strength of the histogram provided by MegAlign program (version 8.0.2) (ranging 0 to 100%); six levels of homology are determined with the highest=100%, decreasing in 20% increments to the lowest (0%) and shown by bar height in FIG. 2. A score of 100 is the highest conservation and indicates there are no changes at that residue when the sequence from human serum albumin is compared with other mammalian albumin sequence, whereas a score of 0 indicates the lowest level of conservation between the aligned sequences.

[0242] Similarly, the homology level score for each amino acid residue in HSA (FIG. 1, column labelled `Align 2 (Var. Sps. (`various species (i.e. mammalian and non-mammalian), Clustal V`)) calculated using the strength of the histogram provided by Megalign when various albumins (including non-mammalian albumins) are aligned using Clustal V. A person skilled in the art will appreciate that a range of different albumin sequences and alignment algorithms may be used to calculate the homology level score.

[0243] When using homology scores, such as those described in reference to the alignments of FIGS. 2 and 3, to identify candidate residues for the present invention, preferred residues include those which are not highly conserved (for example those with a score of less than 40, more preferably less than 20 and most preferably 0) are preferred and those with a higher level of homology (for example those with a score of more than 40, more than 60, more than 80 and most preferably 100) are less preferred.

[0244] Each of the amino acid residue in HSA (SEQ ID No. 1) were scored according to whether the adjacent residue was highly (100%) conserved when HSA is aligned with mammalian albumins (FIG. 1, column labelled `Adj. 100%'s (Align 1). This is because if an amino acid is within a highly conserved domain, it may be important to the structure of function of the protein and thus disruption may be undesirable. In FIG. 1, a score of 0 indicates the residue is not adjacent to any residue with 100% homology (with reference to the alignment of FIG. 2) when HSA is aligned with mammalian albumins; a score of 1 indicates that the residue is adjacent to one residue with 100% homology when HSA is aligned with mammalian albumins; a score of 2 indicates that the residue is adjacent to two residues with 100% homology when HSA is aligned with mammalian albumins. Residues with a score of 0 or 1 are preferred. Residues with a score of 0 are most preferred.

[0245] For example, amino acid residues with a score of 2 (such as valine-7 (V7)) are preferably deselected using the method of the invention since these amino acid residues were assumed to occur in a region of high homology which would be unlikely to accept a mutation to an alternative amino acid. Similarly, phenylalanine-11 (F11) is adjacent to one 100% conserved residue, in a region of conserved residues, and is less preferred to a residue, such as alanine-2 (A2), which has no adjacent 100% conserved residues.

[0246] In accordance with the invention, additional information such as preferred sites for insertion, deletions or substitutions may also be obtained by alignment analysis. For example, mouse albumin contains 36 cysteine residues, all the cysteines involved in disulphide bonding (by homology to HSA) are present, as is cysteine-34, however a cysteine residue is present at 579 on mature mouse protein but not other mammalian albumin sequences therefore thio-albumin mutein S579C may be preferred as its lack of homology with other mammalian albumins suggests that it may not be particularly important to the structure and/or function of this albumin.

[0247] In addition, using the alignments of various mammalian albumin family and Clustal W (FIG. 2) shows that compared to other mammalian albumins, gerbil albumin has an additional alanine residue between alanine-2 (A2) and histidine-3 (H3), indicating that insertion of a cysteine residues after residue 2 (e.g. A2 of SEQ ID No. 1) and before residue 3 (e.g. H3 of SEQ ID No. 1) is preferred.

[0248] Compared to other mammalian albumins, guinea pig albumin has a serine residue at cysteine-34 (C34). In examples where deletion of the free thiol group at cysteine-34 is required, a mutation such as C34S may be preferred.

[0249] Most mammalian albumin sequences (with the exception of human serum albumin) have a sequence which is less than or equal to 584 amino acids in length (less than or equal to 608 amino acids including leader sequence). Using the alignment in FIG. 2 the additional amino acid residue present on human serum albumin appears to be at the C-terminus without any cognate alignment amino acid residues in the other mammalian serum albumin sequences. Thus a thio-albumin variant containing G584C and a deletion of L585 may be preferred.

[0250] A number of albumin sequences (Bovine, Donkey, Goat, Horse, Sheep, Pig)are 583 amino acids in length (607 amino acids including leader sequence). Using the alignment in FIG. 2, it can be seen that these species albumin sequences do not have a residue corresponding R117 (R141 including leader sequence) therefore a thio-albumin containing V116C and a deletion of R117 or a thio-albumin containing a deletion of R117 and P118C may be preferred. In such a thio-albumin the length of the amino acid sequence would be reduced relative to SEQ ID No. 1.

[0251] The alignment used in FIG. 2 and the conclusions drawn are in particular with reference to mammalian albumins and Clustal W, the skilled person will appreciate that the teaching applies likewise to other members of the albumin family and to alternative alignment algorithms.

Alignment and Identity

[0252] The albumin variant may have at least 40% identity with SEQ ID NO: 1, particularly at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity.

[0253] Identity may be calculated using any method, for example those described herein.

Conjugation

[0254] The thio-albumin may optionally be fused to one or more conjugation partners for example through a genetic or chemical fusion. For a thio-albumin which comprises a genetic fusion, the fusion may be at the N- or C-terminus or comprise an insertion.

[0255] With respect to genetic fusions of albumin, the skilled person will also appreciate that the open reading frame of any other gene or variant, or part or either, can be utilised as an open reading frame for use with the present invention. For example, the open reading frame may encode a protein comprising any sequence, be it a natural protein (including a zymogen), or a variant, or a fragment (which may, for example, be a domain) of a natural protein; or a totally synthetic protein; or a single or multiple fusion of different proteins (natural or synthetic). Such proteins can be taken, but not exclusively, from the lists provided in WO 01/79258, WO 01/79271, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, or a variant or fragment thereof; the disclosures of which are incorporated herein by reference. Although these patent applications present the list of proteins in the context of fusion partners for albumin, the present invention is not so limited and, for the purposes of the present invention, any of the proteins listed therein may be presented alone or as fusion partners for albumin or any other protein or fragment or variant of any of the above, as a desired polypeptide. Examples of chemical fusions (also known as conjugations) of albumin are given in Leger et al. (2004) Bioorg Med Chem Lett 14(17): 4395-8; and Thibaudeau et al. (2005). Bioconjug Chem 16(4): 1000-8.

[0256] An advantage of using a genetically or chemically fused albumin is that either or all of the molecules which contribute to the fusion may have improved properties relative to the unfused molecule(s) (Balan et al. (2006) Antivir Ther 11(1): 35-45). Albumins and albumin particles are also important for carrying and delivering drugs and prodrugs to their sites of action (Kratz, F. (2008) Journal of Controlled Release, 132 (3), p.171-183). Fusion and particle technologies offer improved dosing regimes due to improved pharmacokinetic properties, such as half-life extension, and may improve bioavailability and protect the fused conjugation partner, for example bioactive molecule, from inactivation.

[0257] The polypeptide may display modified (e.g. reduced) glycosylation, such as, but not limited to reduced N-linked glycosylation or reduced O-linked glycosylation. The N-linked glycosylation pattern of an albumin molecule can be modified by adding/removing amino acid glycosylation consensus sequences such as N-X-S/T, at any or all of the N, X, or S/T position. Albumin polymorphisms exist with N-linked glycosylation. Albumin mutants may have recycling time such that the efficacy of a mutant as a bioactive carrier is improved. Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell. For example, the albumin protein sequence of SEQ ID No. 1 does not contain any sites for N-linked glycosylation and has not been reported to be modified, in nature, by O-linked glycosylation. However, it has been found that recombinant human albumin ("rHA") produced in a number of yeast species can be modified by O-linked glycosylation, generally involving mannose. The mannosylated albumin is able to bind to the lectin Concanavalin A. The amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced. Alternatively, the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.

[0258] If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia pastoris or Kluyveromyces lactis. The sequence of PMT1 (or any other PMT gene) isolated from S. cerevisiae may be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species. The cloning of the PMT1 homologue of Kluyveromyces lactis is described in WO 94/04687.

[0259] The step of "purifying the thus expressed heterologous protein from the cultured host cell or the culture medium" optionally comprises cell immobilization, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilization, separation and/or breakage.

[0260] Cell immobilization techniques, such as encasing the cells using calcium alginate beads, are well known in the art. Similarly, cell separation techniques, such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like are well known in the art. Likewise, methods of cell breakage, including beadmilling, sonication, enzymatic exposure and the like are well known in the art.

[0261] The at least one other purification step may be any other step suitable for protein purification known in the art. For example purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference.

Production of Conjugation Competent Albumin ("Thio-Albumin")

[0262] The thio-albumin or fusions of thio-albumin and another protein or proteins can be prepared by methods know to the art (Sanker, (2004), Genetic Eng. News, 24, 22-28, Schmidt, (2004), Appl. Microbiol. Biotechnol., 65, 363-372) including but not limited to expression in mammalian cell culture (Mason et al., (2004), Protein Expr. Purif., 36, 318-326; Mason at al., (2002), Biochemistry, 41, 9448-9454) from cells lines such as CHO (and its variants), NS0, BHK, HEK293, Vero or PERC6 cells by transformation or transient expression; insect cell culture (Lim et al., (2004) Biotechnol. Prog., 20, 1192-1197); plant cell culture from such plants as Lemna or Oryza sativa; transgenic animals (Dyck et al., (2003)

[0263] Trends in Biotechnology, 21, 394-399); transgenic plants (Ma et al., (2003) Nature Reviews Genetics, 4, 794-805); Gram positive and Gram negative bacteria such as Bacillus and Escherichia coli (Steinlein, and Ikeda, (1993), Enzyme Microb. Technol., 15, 193-199); filamentous fungi including but not restricted to Aspergillus spp (EP 238023, U.S. Pat. No. 5,364,770, U.S. Pat. No. 5,578,463, EP184438, EP284603, WO 2000/056900, WO9614413), Trichoderma spp and Fusarium spp (Navalainen at al., (2005), Trends in Biotechnology, 23, 468-473).

[0264] The host cell may be any type of cell. The host cell may or may not be an animal (such as mammalian, avian, insect, etc.), plant (such as Oryza sativa), fungal or bacterial cell. Bacterial and fungal, such as yeast, host cells may or may not be preferred.

[0265] Typical prokaryotic vector plasmids are: pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, N.J., USA); pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, pNH46A available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA).

[0266] A typical mammalian cell vector plasmid is pSVL available from Pharmacia (Piscataway, N.J., USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, N.J., USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.

[0267] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence and, for example appropriate transcriptional or translational controls. One such method involves ligation via cohesive ends. Compatible cohesive ends can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.

[0268] A further method uses synthetic double stranded oligonucleotide linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3' ends. Synthetic linkers and pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end. Alternatively a DNA fragment or DNA fragments can be ligated together by the action of DNA ligase in the presence or absence of one or more synthetic double stranded oligonucleotides optionally containing cohesive ends.

[0269] Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Sigma-Genosys Ltd, London Road, Pampisford, Cambridge, United Kingdom.

[0270] The thio-albumin or fusions of thio-albumin and another protein or proteins may be expressed from a nucleotide sequence, which may or may not contain one or more introns. Additionally the nucleotide sequence may or may not be codon optimised for the host by methods known to the art.

[0271] The thio-albumin or fusions of thio-albumin and another protein or proteins can be expressed as variants with reduced N-linked glycosylation. Accordingly, in case of human serum albumin (HSA), it may be particularly advantageous to use a yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins, for instance by disruption of the gene coding sequence. Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell. The mannosylated albumin would be able to bind to the lectin Concanavalin A. The amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may or may not be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced. Alternatively, the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody. Alternatively, the yeast could be cultured in the presence of a compound that inhibits the activity of one of the PMT genes (Duffy et al, "Inhibition of protein mannosyltransferase 1 (PMT1) activity in the pathogenic yeast Candida albicans", International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, 26-31 August 2001, Monte Verita, Switzerland, Poster Abstract P38; the poster abstract may be viewed at http://www.micro.biol.ethz.ch/cellwall/). If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia pastoris or Kluyveromyces lactis. The sequence of PMT1 (or any other PMT gene) isolated from S. cerevisiae may be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species. The cloning of the PMT1 homologue of Kluyveromyces lactis is described in WO 94/04687.

[0272] The yeast may or may not also have a deletion of the HSP150 and/or YAP3 genes as taught respectively in WO 95/33833 and WO 95/23857.

[0273] The HSA variant may be produced by recombinant expression and secretion. Where the expression system (i.e. the host cell) is yeast, such as Saccharomyces cerevisiae, suitable promoters for S. cerevisiae include those associated with the PGK1 gene, GAL1 or GAL10 genes, TEF1, TEF2, PYK1, PMA1, CYC1, PHO5, TRP1, ADH1, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, a-mating factor pheromone, a-mating factor pheromone, the PRB1 promoter, the PRA1 promoter, the GPD1 promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).

[0274] Suitable transcription termination signals are well known in the art. Where the host cell is eukaryotic, the transcription termination signal is preferably derived from the 3' flanking sequence of a eukaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different. In that case, and where the host is a yeast, preferably S. cerevisiae, then the termination signal of the S. cerevisiae ADH1, ADH2, CYC1, or PGK1 genes are preferred.

[0275] It may be beneficial for the promoter and open reading frame of the gene encoding the recombinant protein comprising the sequence of an albumin mutant to be flanked by transcription termination sequences so that the transcription termination sequences are located both upstream and downstream of the promoter and open reading frame, in order to prevent transcriptional read-through into any neighbouring genes, such as 2 .mu.m genes, and vice versa.

[0276] In one embodiment, the favoured regulatory sequences in yeast, such as Saccharomyces cerevisiae, include: a yeast promoter (e.g. the Saccharomyces cerevisiae PRB1 promoter), as taught in EP 431 880; and a transcription terminator, preferably the terminator from Saccharomyces ADH1, as taught in EP 60 057.

[0277] It may be beneficial for the non-coding region to incorporate more than one DNA sequence encoding a translational stop codon, such as UAA, UAG or UGA, in order to minimise translational read-through and thus avoid the production of elongated, non-natural fusion proteins. The translation stop codon UAA is preferred.

[0278] In one preferred embodiment, the recombinant protein comprising the sequence of an albumin mutant is secreted. In that case, a sequence encoding a secretion leader sequence may be included in the open reading frame. Thus, a polynucleotide according to the present invention may comprise a sequence that encodes a recombinant protein comprising the sequence of an albumin mutant operably linked to a polynucleotide sequence that encodes a secretion leader sequence. Leader sequences are usually, although not necessarily, located at the N-terminus of the primary translation product of an ORF and are generally, although not necessarily, cleaved off the protein during the secretion process, to yield the "mature" protein. Thus, in one embodiment, the term "operably linked" in the context of leader sequences includes the meaning that the sequence that encodes a recombinant protein comprising the sequence of an albumin mutant is linked, at its 5' end, and in-frame, to the 3' end of a polynucleotide sequence that encodes a secretion leader sequence. Alternatively, the polynucleotide sequence that encodes a secretion leader sequence may be located, in-frame, within the coding sequence of the recombinant protein comprising the sequence of an albumin mutant, or at the 3' end of the coding sequence of the recombinant protein comprising the sequence of an albumin mutant.

[0279] Numerous natural or artificial polypeptide leader sequences (also called secretion pre regions and pre/pro regions) have been used or developed for secreting proteins from host cells. Leader sequences direct a nascent protein towards the machinery of the cell that exports proteins from the cell into the surrounding medium or, in some cases, into the periplasmic space.

[0280] For production of proteins in eukaryotic species such as the yeasts Saccharomyces cerevisiae, Zygosaccharomyces species, Kluyveromyces lactis and Pichia pastoris, a secretion leader sequence may be used. This may comprise a signal (pre) sequence or a prepro leader sequence. Signal sequences are known to be heterogeneous in their amino acid sequence (Nothwehr and Gordon 1990, Bioessays 12, 479-484, or Gierasch 1989, Biochemistry 28, p923-930). In essence, signal sequences are generally N-terminally located, have a basic n-region, a hydrophobic h-region and a polar c-region. As long as this structure is retained the signal sequence will work, irrespective of the amino acid composition. How well they work, i.e. how much mature protein is secreted, depends upon the amino acid sequence. Accordingly, the term "signal peptide" is understood to mean a presequence which is predominantly hydrophobic in nature and present as an N-terminal sequence of the precursor form of an extracellular protein expressed in yeast. The function of the signal peptide is to allow the expressed protein to be secreted to enter the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. Known leader sequences include those from the S. cerevisiae acid phosphatase protein (Pho5p) (see EP 366 400), the invertase protein (Suc2p) (see Smith et aL (1985) Science, 229, 1219-1224) and heat-shock protein-150 (Hsp150p) (see WO 95/33833). Additionally, leader sequences from the S. cerevisiae mating factor alpha-1 protein (MF.alpha.-1) and from the human lysozyme and human serum albumin (HSA) protein have been used, the latter having been used especially, although not exclusively, for secreting human albumin. WO 90/01063 discloses a fusion of the MF.alpha.-1 and HSA leader sequences (also known as the fusion leader sequence (FL)). In addition, the natural albumin leader sequence may or may not be used to direct secretion of the recombinant protein comprising the sequence of an albumin mutant.

[0281] The skilled person will appreciate that any suitable plasmid may be used, such as a centromeric plasmid. The examples provide suitable plasmids (centromeric YCplac33-based vectors) for use to transform yeast host cells of the present invention. Alternatively, any other suitable plasmid may be used, such as a yeast-compatible 2.sub.pm-based plasmid.

[0282] Plasmids obtained from one yeast type can be maintained in other yeast types (Irie et al, 1991, Gene, 108(1), 139-144; Irie et al, 1991, Mol. Gen. Genet., 225(2), 257-265). For example, pSR1 from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae. In one embodiment the plasmid may or may not be a 2 .mu.m-family plasmid and the host cell will be compatible with the 2 .mu.m-family plasmid used (see below for a full description of the following plasmids). For example, where the plasmid is based on pSR1, pSB3 or pSB4 then a suitable yeast cell is Zygosaccharomyces rouxii; where the plasmid is based on pSB1 or pSB2 then a suitable yeast cell is Zygosaccharomyces bailli; where the plasmid is based on pSM1 then a suitable yeast cell is Zygosaccharomyces fermentati; where the plasmid is based on pKD1 then a suitable yeast cell is Kluyveromyces drosophilarum; where the plasmid is based on pPM1 then a suitable yeast cell is Pichia membranaefaciens; where the plasmid is based on the 2 .mu.m plasmid then a suitable yeast cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Thus, the plasmid may be based on the 2 .mu.m plasmid and the yeast cell may be Saccharomyces cerevisiae. A 2 .mu.m-family plasmid can be said to be "based on" a naturally occurring plasmid if it comprises one, two or preferably three of the genes FLP, REP1 and REP2 having sequences derived from that naturally occurring plasmid.

[0283] Useful yeast episomal plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems (La Jolla, Calif. 92037, USA), YEp24 (Botstein, D., et al. (1979) Gene 8, 17-24), and YEplac122, YEplac195 and YEplac181 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534). Other yeast plasmids are described in WO 90/01063 and EP 424 117, as well as the "disintegration vectors of EP-A-286 424 and WO2005061719. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3, as are Ylplac204, Ylplac211 and Ylplac128 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534). Plasmids pRS413-416 are Yeast Centromere plasmids (YCps) as are YCplac22, YCplac33 and YCplac111 (Gietz, R. D. and Sugino. A. (1988) Gene 74, 527-534).

[0284] Where one or more of the helper (also known as `chaperone`) protein(s) and/or protein product of choice are encoded by a plasmid-borne polynucleotide sequence, the host cell type may be selected for compatibility with the plasmid type being used. Such plasmids are disclosed in WO2005061719. Preferred helper proteins include PDI1, AHAI, ATP11, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNS1, CPR3, CPRE, DER1, DER3, DOA4, ERO1, EUG1, ERV2, EPS1, FKB2, FMO1, HCH1, HRD3, HSP10, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, KAR2, JEM1, MDJ1, MDJ2, MPD1, MPD2, PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2, LHS1, MGE1, MRS11, NOB1, ECM10, SCJ1, SSA1, SSA2, SSA3, SSA4, SSBI, SSB2, SSC1, SSE2, SIL1, SLS1, ORM1, ORM2, PERI, PTC2, PSE1, UBC7, UBI4 and HAC1 or a truncated intronless HAC1 (Valkonen et al. 2003, Applied Environ. Micro., 69, 2065). Such helper proteins are disclosed in WO 2005/061718, WO 2006/067511 and WO 2006/136831.

[0285] Plasmids as defined herein may be introduced into a host through standard techniques. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3.sup.rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. Methods for the transformation of S. cerevisiae are taught generally in EP 251 744, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA.

[0286] Electroporation is also useful for transforming cells and is well known in the art for transforming fungal (including yeast) cell, plant cells, bacterial cells and animal (including vertebrate) cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

[0287] Generally, the plasmid will transform not all of the hosts and it will therefore be necessary to select for transformed host cells. Thus, a plasmid may comprise a selectable marker, including but not limited to bacterial selectable marker and/or a yeast selectable marker. A typical bacterial selectable marker is the .beta.-lactamase gene although many others are known in the art. Typical yeast selectable marker include LEU2, TRP1, HIS3, HIS4, URA3, URA5, SFA 1, ADE2, MET15, LYS5, LYS2, ILV2, FBA1, PSE1, PDI1 and PGK1. Those skilled in the art will appreciate that any gene whose chromosomal deletion or inactivation results in an unviable host, so called essential genes, can be used as a selective marker if a functional gene is provided on the plasmid, as demonstrated for PGK1 in a pgk1 yeast strain (Piper and Curran, 1990, Curr. Genet. 17, 119). Suitable essential genes can be found within the Stanford Genome Database (SGD), (http:://db.yeastgenome.org). Any essential gene product (e.g. PDI1, PSE1, PGK1 or FBA1) which, when deleted or inactivated, does not result in an auxotrophic (biosynthetic) requirement, can be used as a selectable marker on a plasmid in a host cell that, in the absence of the plasmid, is unable to produce that gene product, to achieve increased plasmid stability without the disadvantage of requiring the cell to be cultured under specific selective conditions. By "auxotrophic (biosynthetic) requirement" we include a deficiency which can be complemented by additions or modifications to the growth medium. Therefore, preferred "essential marker genes" in the context of the present application are those that, when deleted or inactivated in a host cell, result in a deficiency which cannot be complemented by additions or modifications to the growth medium. Additionally, a plasmid may comprise more than one selectable marker.

[0288] Transformed host cells may be cultured for a sufficient time and under appropriate conditions known to those skilled in the art, and in view of the teachings disclosed herein, to permit the expression of the helper protein(s) and the protein product of choice.

[0289] The culture medium may be non-selective or place a selective pressure on the maintenance of a plasmid.

[0290] Methods for culturing prokaryotic host cells, such as E. coli, and eukaryotic host cells, such as mammalian cells are well known in the art. Methods for culturing yeast are generally taught in EP 330 451 and EP 361 991.

[0291] The thus produced protein product of choice may be present intracellularly or, if secreted, in the culture medium and/or periplasmic space of the host cell.

Preparation of a Polypeptide

[0292] The step of "purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium" optionally comprises cell immobilisation, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilisation, separation and/or breakage.

[0293] Thio-albumin of the invention may be purified from the culture medium by any technique that has been found to be useful for purifying such proteins. Similarly, cell separation techniques, such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like) are well known in the art. Likewise, methods of cell breakage, including beadmilling, sonication, enzymatic exposure and the like are well known in the art.

[0294] The "at least one other purification step" may be any other step suitable for protein purification known in the art. For example purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, U.S. Pat. No. 5,728,553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference. Suitable methods include ammonium sulphate or ethanol precipitation, acid or solvent extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography ("HPLC"), reverse phase HPLC, conductivity adjustment and the like.

[0295] The polypeptide may be purified to a commercially or industrially acceptable level of purity. By commercially or industrially acceptable level of purity, we include the provision of the thio-albumin and/or thio-albumin-conjugate in which other material (for example, one or more contaminants) are present at a level of less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% and, most preferably at a level of 0%.

[0296] A commercially or industrially acceptable level of purity may be obtained by a relatively crude purification method by which the protein product of choice is put into a form suitable for its intended purpose. A protein preparation that has been purified to a commercially or industrially acceptable level of purity may, in addition to the protein product of choice, also comprise, for example, cell culture components such as host cells or debris derived therefrom. Alternatively, high molecular weight components (such as host cells or debris derived therefrom) may or may not be removed (such as by filtration or centrifugation) to obtain a composition comprising the protein product of choice and, optionally, a functionally acceptable level of low molecular weight contaminants derived from the cell culture process.

[0297] The protein may or may not be purified to achieve a pharmaceutically acceptable level of purity. A protein has a pharmaceutically acceptable level of purity if it is essentially pyrogen free and can be used for its intended purpose and hence be administered in a pharmaceutically efficacious amount without causing medical effects not associated with the activity of the protein.

[0298] The thio-albumin and/or thio-albumin-conjugate may be provided at a concentration of at least 10.sup.-4 g.L.sup.-1, 10.sup.-3 g.L.sup.-1, 0.01 g.L.sup.-1, 0.02 g.L.sup.-1, 0.03 g.L.sup.-1, 0.04 g.L.sup.-1, 0.05 g.L.sup.-1, 0.06 g.L.sup.-1, 0.07 g.L.sup.-1, 0.08 g.L.sup.-1, 0.09 g.L.sup.-1, 0.1 g.L.sup.-1, 0.2 g.L.sup.-l, 0.3 g.L.sup.-1, 0.4 g.L.sup.-1, 0.5 g.L.sup.-1, 0.6 g/L.sup.-1, 0.7 g.L.sup.-1, 0.8 g.L.sup.-1, 0.9 0 g.L.sup.-1, 1 g.L.sup.-1, 2 g.L.sup.-1, 3 g.L.sup.-1, 4 g.L.sup.-1, 5 g.L.sup.-1, 6 g.L.sup.-1, 7 g.L.sup.-1, 8 g.L.sup.-1, 9 g.L.sup.-1, 10 g.L.sup.-1, 15 g.L.sup.-1, 20 g.L.sup.-1, 25 g.L.sup.-1, 30 g.L.sup.-1, 40 g.L.sup.-1, 50 g.L.sup.-1, 60 g.L.sup.-1, 70 g.L.sup.-1, 70 g.L.sup.-1, 90 gL.sup.-1, 100 g.L.sup.-1, 150 g.L.sup.-1, 200 g.L.sup.-1, 250 g.L.sup.-1, 300 g.L.sup.-1, 350 g.L.sup.-1, 400 g.L.sup.-1, 500 g.L.sup.-1, 600 g.L.sup.-1, 700 g.L.sup.-1, 800 g.L.sup.-1, 900 g.L.sup.-1, 1000 g.L.sup.-1.

[0299] A method of the present invention may or may not further comprise the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.

[0300] Although it is possible for a therapeutically useful protein obtained by a process of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers or diluents. The carrier(s) or diluent(s) must be "acceptable" in the sense of being compatible with the desired protein. Typically, the carriers or diluents will be water or saline which will be sterile and pyrogen free.

[0301] Alternatively, a method of the present invention may or may not further comprise the step of lyophilising the thus purified protein product of choice.

[0302] Formulation of Thio-Albumin or Conjugate

[0303] The thio-albumin may be formulated by strategies given in "Protein Formulation and Delivery", E. J. McNally (Ed.), published by Marcel Dekker Inc. New York 2000 and "Rational Design of Stable Protein Formulations-Theory and Practice"; J. F. Carpenter and M. C. Manning (Ed.) Pharmaceutical Biotechnology Vol 13. Kluwer Academic/Plenum Publishers, New York 2002, Yazdi and Murphy, (1994) Cancer Research 54, 6387-6394, Widera et al., (2003) Pharmaceutical Research 20, 1231-1238; Lee et al., (2005) Arch. Pharm. Res. 28, 722-729. Examples of formulation methods are as follows:

[0304] Method #1: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4.degree. C., -20.degree. C. or -80.degree. C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl.

[0305] Method #2: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4.degree. C., -20.degree. C. or -80.degree. C. in 0.01 M-0.1 M phosphate buffered saline (pH 7.0-8.0) containing 0.01 M-0.2 M NaCl and containing 10-20 mg/L Polysorbate 80.

[0306] Method #3: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4.degree. C., -20.degree. C. or -80.degree. C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0).

[0307] Method #4: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be stored at 4.degree. C., -20.degree. C. or -80.degree. C. in 0.01 M-0.2 M NaCl (pH 7.0-8.0) containing 10-20 mg/L Polysorbate 80.

Freeze-Dried Formulations

[0308] Method #5: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against water, freeze dried and stored at 4.degree. C., -20.degree. C. or -80.degree. C.

[0309] Method #6: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be dialysed against 0.01 M-0.2 M NaCl (pH 7.0-8.0), freeze dried and stored at 4.degree. C., -20.degree. C. or -80.degree. C.

Nanoparticle Formulation

[0310] The thio-albumin of the invention (and/or its conjugated form) may be used to produce nanoparticles and/or be entrapped within a nanoparticle or liposome.

[0311] The thio-albumin of the invention may be used with and/or in and/or as a nanoparticle and/or liposome. A problem of current conjugation strategies is maintaining both the pharmacological and immunological activity of the conjugation partner, such as a bioactive-targeting ligand conjugate. There is likely to be a maximum number of protein targeting ligand/bioactive moieties (conjugation partners) possible for conjugation to a protein and if this number is exceeded the targeting ligand does not retain its biological activity. Preferably the biological activity of the conjugation partner is not reduced by conjugation to an albumin of the invention.

[0312] Liposomes and nanoparticles may be used to entrap bioactive compounds. They provide a mechanism for enhanced delivery of drugs such as bioactive compounds, or uptake by target cells and/or a reduction in the toxicity of the free bioactive to non-target organs which may result in an increased therapeutic index and/or reduced side effects. In addition, many solvent-based formulations required for the delivery of some bioactive compounds (e.g. taxanes) are associated with toxicity which limits the maximum dose which can be given to a patient. Liposome and nanoparticle delivery may also be advantageous for such bioactive compounds, since they would allow larger amounts of the bioactive compound to be delivered whilst avoiding some of the toxicities of solvent-based formulations (Hawkins et al (2008) Advanced Drug Delivery Reviews, 60, 8, p 876-885).

[0313] Methods for attaching targeting ligands to liposomes and nanoparticles are known in the art (reviewed in Nobs et al (2004) Journal of Pharmaceutical Sciences Vol 93 p 980-1992) and may be used in accordance with the invention. Attachment methods may be non-covalent or covalent. Covalent reactions appear to be favourable, because covalent linkage is more stable than noncovalent methods. Lipids for the covalent or non-covalent attachment of proteins, peptides, or drugs to the liposome surface are available commercially (for example Avanti Polar Lipids Inc Alabaster, Ala., USA). There are 3 major classes of functionality: conjugation through disulphide or thioether formation, amide bond formation, or biotin/streptavidin binding, any of these may be used in the invention.

[0314] A number of methods relying on covalent coupling ligands to the surface of liposomes via thioether bonds have been described, most commonly utilizing the highly efficient reaction of maleimide with thiol groups. Functionalized lipid anchors commonly added to liposomes, and which may be used in or with the invention, include, but are not limited those containing maleimide such as N-[4-(p-maleimidophenyl) butyramide]-PE (N-MPB]-PE) or N-[4-(p-maleimidomethyl) cyclohexane-carboxamide) (MCC-PE) which allow convenient covalent coupling of the targeting moiety via a stable thioether bond (Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288).

[0315] Method #7: Following purification the free thiol containing albumin mutein of the invention or the conjugate can be formulated into nanoparticles prepared according to known procedures for preparing nanoparticles, such as procedures disclosed in WO 2004/071536 A1 and WO 2008/007146 A1, both incorporated herein by reference.

[0316] Similarly materials for the formation of nanoparticles, including but are limited to Poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and COOH-PLA are commercially available and may be functionalized with maleimide or other known chemistries according to known literature for nanoparticle formation. Any of these may be used in or with the invention.

[0317] Another convenient way for covalent coupling of ligands to liposomes involves conjugation of two thiols to form a disulphide; however under the reductive conditions in serum more stable conjugation chemistries involving one free thiol group may be preferred. Chemistries such as (PDP-PE) allow covalent coupling via a disulphide bond. Modification of the ligand to introduce a free thiol group or a functionalized linker may be used. An advantage of the thio-albumin of the invention is that no ligand modification is required. However, ligand modification may optionally be used in addition to the invention.

[0318] Frequently thiol groups are not present in proteins, or are not present in sufficient amounts or at the desired location. Thus, most cases of covalent coupling of one of more ligands to a liposome via thioether or disulphide bonds requires the use of heterobifunctional cross linking agents (described herein with reference to conjugation). Some heterobifunctional cross linking agents (such as SPDP and SATA) require a de-protection step. The thio-albumin of the invention overcomes the requirement for this additional processing.

[0319] Alternatively thio-albumin could be conjugated to liposomes or nanoparticles by other chemistries, known to the art. For example, thio-albumin could be attached by an amide bond using a functionalised lipid anchor with either amine or carboxyl functional groups (examples include DSPE-PEG-COOH) which reacts with the primary amine of the ligand. Direct cross linking between primary amines and the surface of liposomes may also be used. The one or more free thiol groups of thio-albumin would then be available for conjugation to another conjugation partner.

[0320] Following conjugation, a conjugation partner (e.g. bioactive molecule) may show a reduction in its activity (e.g. bioactivity). Thio-albumin described in this invention may overcome this problem by providing a conjugate, nanoparticle and/or liposome in which the conjugation partner is located and/or orientated with respect to a thio-albumin such that the conjugation partner retains at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of its unconjugated activity.

Conjugation Partner

[0321] The term `conjugation partner` includes bioactive agents, imaging agents, diagnostic agents, contrast agents and therapeutic compounds such as chemotherapeutic drugs and radiopharmaceuticals. A thio-albumin of the invention may be conjugated to one or more conjugation partners.

Imaging Agents, Diagnostic Compounds, Contrast Agents and Therapeutic Compounds

[0322] The use of diagnostic agents, imaging agents and biological "contrast" agents are well known to the art. A diagnostic agent is any pharmaceutical product used as part of a diagnostic test (i.e. together with the equipment and procedures that are needed to assess the test result). The diagnostic agent may be used in vivo, ex vivo or in vitro.

[0323] The ability of albumin to accumulate in damaged muscle fibres of dystrophic muscle has been well described. For example, a Gadolinium-DTPA-albumin conjugate may be used as a combined diagnostic and therapeutic tool to visualize and monitor, for example, dystrophic muscle by magnetic resonance imaging (MRI) and for the delivery of putative therapeutics bound to albumin for effective targeting to dystrophic muscle (Amthor et al. (2004) Neuromuscular Disorders 14912: 791-796). Malignant tumours often show an increased uptake and metabolism of albumin. The use of gadolinium-albumin conjugate has also been described for improved imaging of malignant tumours and to determine by MRI tumours sensitive to a therapy with drug-conjugated albumin (Kiessling et al. (2002) Investigative Radiology 37(4): 93-198).

[0324] Current imaging agents often degrade quickly whilst longer-lasting agents are often toxic. The use of albumin conjugates may be especially useful to increase the half life of imaging agents and would therefore permit imaging over an extended period of time. WO2005/082423 describes the use of serum albumin conjugated to fluorescent substances for imaging.

[0325] A thio-albumin of this invention may be conjugated to two or more molecules selected from imaging agents, diagnostic agents, therapeutic compounds and contrast agents.

[0326] Tumours (and muscle degeneration) show enhanced uptake of albumin (EPR: Enhanced Permeation and Retention). Albumin conjugates may be used for enhanced imaging, and also to assess whether tumours (or or other tissues and organs) would be suitable for albumin conjugated drugs.

Bioactive Compound

[0327] The bioactive compound may be a therapeutic or diagnostic compound. The therapeutic compound may be a chemotherapy drug for use in cancer chemotherapy. It may be cytostatic or cytotoxic; it may be a tumor-inhibiting agent.

[0328] The bioactive compound may already contain a free thiol group, e.g. a polypeptide containing a Cysteine residue with a free thiol group. Alternatively, the bioactive compound may be modified so as to contain a free thiol group. Thus, the amino acid sequence of a polypeptide may be altered so as to include a Cysteine residue with a free thiol group, or the bioactive compound may be chemically derivatized to include a free thiol group.

[0329] The bioactive compound may be a polypeptide (protein), particularly a recombinant protein pharmaceutical. It may be a chemotherapy or radiotherapy drug used to treat cancers and other related diseases.

[0330] The free thiol containing albumin mutein of the invention (thio-albumin) can be conjugated via the free thiol group, or groups if the albumin mutein of the invention contains more than one free thiol, to at least one bioactive compound by methods know to the art. The bioactive compound includes but is not limited to, peptides, polypeptides or proteins (either natural, recombinant, or synthetic) (Debinski, (2002) Cancer Investigation 20, 801-809, O'Keefe and Draper et al., (1985) JBC 260, 932-937, Xia et al., (2000) J. Pharmacology Experimental Therapeutics 295, 594-600, Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384, Humphries, et al., (1994) J. Tissue Culture Methods 16, 239-242, Wenning et al., (1998) Biotech. Bioeng. 57, 484-496, Yazdi and Murphy, (1994) Cancer Research 54, 6387-6394, Weaver and Laske (2003) J. Neuro-Oncology 65, 3-13, Widera at al., (2003) Pharmaceutical Research 20, 1231-1238, Daniels, T. R. et al. (2006) Clinical Immunology 121, 159-176 and the references included therein); therapeutic and diagnostic drugs or compounds (Mishra et al., (2006) J. Drug Targeting 14, 45-53, Lim and Shen, (2004) Pharmaceutical Research 21, 1985-1992, Fritzer et al., (1996) Biochemical Pharmacology 51, 489-493, Lubgan and Jozwiak (2002) Cell. Mol. Biol. Lett. 7, 98, Daniels, T. R. at al. (2006) Clinical Immunology 121, 159-176 and the references included therein); high molecular weight complexes including but not limited to liposomes, viruses and nanoparticles (Mishra at al., (2006) J. Drug Targeting 14, 45-53, Daniels, T. R. at al. (2006) Clinical Immunology 121, 159-176 and the references included therein); nucleic acids and radionuclides, including DNA, RNA (including siRNA) and their analogs (Lee at al., (2005) Arch. Pharm. Res. 28, 722-729, Huang et al., (2007) FASEB J. 21, 1117-1125, Daniels, T. R. et al. (2006) Clinical Immunology 121, 159-176 and the references included therein) and devices (Humphries, et al., (1994) J. Tissue Culture Methods 16, 239-242 and the references included therein). Additionally the entity can itself be modified by methods known to the art.

Therapeutic Compounds

[0331] 4-1BB ligand, 5-helix, A human C-C chemokine, A human L105 chemokine, A human L105 chemokine designated huL105_3., A monokine induced by gamma-interferon (MIG), A partial CXCR4B protein, A platelet basic protein (PBP), .alpha.1-antitrypsin, .quadrature..quadrature.ACRP-30 Homologue; Complement Component C1q C, Adenoid-expressed chemokine (ADEC), aFGF; FGF-1, AGF, AGF Protein, albumin, an etoposide, angiostatin, Anthrax vaccine, Antibodies specific for collapsin, antistasin, Anti-TGF beta family antibodies, antithrombin APM-1; ACRP-30; Famoxin, apo-lipoprotein species, Arylsulfatase B, b57 Protein, BCMA, Beta-thromboglobulin protein (beta-TG), bFGF; FGF2, Blood coagulation factors, BMP Processing Enzyme Furin, BMP-10, BMP-12, BMP-15, BMP-17, BMP-18, BMP-2B, BMP-4, BMP-5, BMP-6, BMP-9, Bone Morphogenic Protein-2, calcitonin, Calpain-10a, Calpain-10b, Calpain-10c, Cancer Vaccine, Carboxypeptidase, C-C chemokine, MCP2, CCR5 variant, CCR7, CCR7, CD11a Mab, CD137; 4-1BB Receptor Protein, CD20 Mab, CD27, CD27L, CD30, CD30 ligand, CD33 immunotoxin, CD40, CD40L, CD52 Mab, Cerebus Protein, Chemokine Eotaxin., Chemokine hIL-8, Chemokine hMCP1, Chemokine hMCP1a, Chemokine hMCP1b, Chemokine hMCP2, Chemokine hMCP3, Chemokine hSDF1b, Chemokine MCP-4, chemokine TECK and TECK variant, Chemokine-like protein IL-8M1 Full-Length and Mature, Chemokine-like protein IL-8M10 Full-Length and Mature, Chemokine-like protein IL-8M3, Chemokine-like protein IL-8M8 Full-Length and Mature, Chemokine-like protein IL-8M9 Full-Length and Mature, Chemokine-like protein PF4-414 Full-Length and Mature, Chemokine-like protein PF4-426 Full-Length and Mature, Chemokine-like protein PF4-M2 Full-Length and Mature, Cholera vaccine, Chondromodulin-like protein, c-kit ligand; SCF; Mast cell growth factor; MGF; Fibrosarcoma-derived stem cell factor, CNTF and fragment thereof (such as CNTFAx15'(Axokine.TM.)), coagulation factors in both pre and active forms, collagens, Complement C5 Mab, Connective tissue activating protein-Ill, CTAA16.88 Mab, CTAP-III, CTLA4-Ig, CTLA-8, CXC3, CXC3, CXCR3; CXC chemokine receptor 3, cyanovirin-N, Darbepoetin, designated exodus, designated huL105_7., DIL-40, Dnase, EDAR, EGF Receptor Mab, ENA-78, Endostatin, Eotaxin, Epithelial neutrophil activating protein-78, EPO receptor; EPOR, erythropoietin (EPO) and EPO mimics, Eutropin, Exodus protein, Factor IX, Factor VII, Factor VIII, Factor X and Factor XIII, FAS Ligand Inhibitory Protein (DcR3), FasL, FasL, FasL, FGF, FGF-12; Fibroblast growth factor homologous factor-1, FGF-15, FGF-16, FGF-18, FGF-3; INT-2, FGF-4; gelonin, HST-1; HBGF-4, FGF-5, FGF-6; Heparin binding secreted transforming factor-2, FGF-8, FGF-9; Glia activating factor, fibrinogen, flt-1, flt-3 ligand, Follicle stimulating hormone Alpha subunit, Follicle stimulating hormone Beta subunit, Follitropin, Fractalkine, fragment. myofibrillar protein Troponin I, FSH, Galactosidase, Galectin-4, G-CSF, GDF-1, Gene therapy, Glioma-derived growth factor, glucagon, glucagon-like peptides, Glucocerebrosidase, glucose oxidase, Glucosidase, Glycodelin-A; Progesterone-associated endometrial protein, GM-CSF, gonadotropin, Granulocyte chemotactic protein-2 (GCP-2), Granulocyte-macrophage colony stimulating factor, growth hormone, Growth related oncogene-alpha (GRO-alpha), Growth related oncogene-beta (GRO-beta), Growth related oncogene-gamma (GRO-gamma), hAPO-4; TROY, hCG, Hepatitus B surface Antigen, Hepatitus B Vaccine, HER2 Receptor Mab, hirudin, HIV gp120, HIV gp41, HIV Inhibitor Peptide, HIV Inhibitor Peptide, HIV Inhibitor Peptide, HIV protease inhibiting peptides, HIV-1 protease inhibitors, HPV vaccine, Human 6CKine protein, Human Act-2 protein, Human adipogenesis inhibitory factor, human B cell stimulating factor-2 receptor, Human beta-chemokine H1305 (MCP-2), Human C-C chemokine DGWCC, Human CC chemokine ELC protein, Human CC type chemokine interleukin C, Human CCC3 protein, Human CCF18 chemokine, Human CC-type chemokine protein designated SLC (secondary lymphoid chemokine), Human chemokine beta-8 short forms, Human chemokine 010, Human chemokine CC-2, Human chemokine CC-3, Human chemokine CCR-2, Human chemokine Ckbeta-7, Human chemokine ENA-78, Human chemokine eotaxin, Human chemokine GRO alpha, Human chemokine GROalpha, Human chemokine GRObeta, Human chemokine HCC-1, Human chemokine HCC-1, Human chemokine I-309, Human chemokine IP-10, Human chemokine L105_3, Human chemokine L105_7, Human chemokine MIG, Human chemokine MIG-beta protein, Human chemokine MIP-1alpha, Human chemokine MIP1beta, Human chemokine MIP-3alpha, Human chemokine MIP-3beta, Human chemokine PF4, Human chemokine protein 331D5, Human chemokine protein 61164, Human chemokine receptor CXCR3, Human chemokine SDF1alpha, Human chemokine SDF1beta, Human chemokine ZSIG-35, Human Chr19Kine protein, Human CKbeta-9, Human CKbeta-9, Human CX3C 111 amino acid chemokine, Human DNAX interleukin-40, Human DVic-1 C-C chemokine, Human EDIRF I protein sequence, Human EDIRF II protein sequence, Human eosinocyte CC type chemokine eotaxin, Human eosinophil-expressed chemokine (EEC), Human fast twitch skeletal muscle troponin C, Human fast twitch skeletal muscle troponin I, Human fast twitch skeletal muscle Troponin subunit C, Human fast twitch skeletal muscle Troponin subunit I Protein, Human fast twitch skeletal muscle Troponin subunit T, Human fast twitch skeletal muscle troponin T, Human foetal spleen expressed chemokine, FSEC, Human GM-CSF receptor, Human gro-alpha chemokine, Human gro-beta chemokine, Human gro-gamma chemokine, Human IL-16 protein, Human IL-1RD10 protein sequence, Human IL-1RD9, Human IL-5 receptor alpha chain, Human IL-6 receptor, Human IL-8 receptor protein hIL8RA, Human IL-8 receptor protein hIL8RB, Human IL-9 receptor protein, Human IL-9 receptor protein variant #3, Human IL-9 receptor protein variant fragment, Human IL-9 receptor protein variant fragment#3, Human interleukin 1 delta, Human Interleukin 10, Human Interleukin 10, Human interleukin 18, Human interleukin 18 derivatives, Human interleukin-1 beta precursor, Human interleukin-1 beta precursor., Human interleukin-1 receptor accessory protein, Human interleukin-1 receptor antagonist beta, Human interleukin-1 type-3 receptor, Human Interleukin-10 (precursor), Human Interleukin-10 (precursor), Human interleukin-11 receptor, Human interleukin-12 40 kD subunit, Human interleukin-12 beta-1 receptor, Human interleukin-12 beta-2 receptor, Human Interleukin-12 p35 protein, Human Interleukin-12 p40 protein, Human interleukin-12 receptor, Human interleukin-13 alpha receptor, Human interleukin-13 beta receptor, Human interleukin-15, Human interleukin-15 receptor from clone P1, Human interleukin-17 receptor, Human interleukin-18 protein (IL-18), Human interleukin-3, human interleukin-3 receptor, Human interleukin-3 variant, Human interleukin-4 receptor, Human interleukin-5, Human interleukin-6, Human interleukin-7, Human interleukin-7., Human interleukin-8 (IL-8), Human intracellular IL-1 receptor antagonist, Human IP-10 and HIV-1 gp120 hypervariable region fusion protein, Human IP-10 and human Muc-1 core epitope (VNT) fusion protein, human liver and activation regulated chemokine (LARC), Human Lkn-1 Full-Length and Mature protein, Human mammary associated chemokine (MACK) protein Full-Length and Mature, Human mature chemokine Ckbeta-7, Human mature gro-alpha, Human mature gro-gamma polypeptide used to treat sepsis, Human MCP-3 and human Muc-1 core epitope (VNT) fusion protein, Human MI10 protein, Human MI1A protein, Human monocyte chemoattractant factor hMCP-1, Human monocyte chemoattractant factor hMCP-3, Human monocyte chemotactic proprotein (MCPP) sequence, Human neurotactin chemokine like domain, Human non-ELR CXC chemokine H174, Human non-ELR CXC chemokine IP10, Human non-ELR CXC chemokine Mig, Human PAI-1 mutants, Human protein with IL-16 activity, Human protein with IL-16 activity, Human secondary lymphoid chemokine (SLC), Human SISD protein, Human STCP-1, Human stromal cell-derived chemokine, SDF-1, Human T cell mixed lymphocyte reaction expressed chemokine (TMEC), Human thymus and activation regulated cytokine (TARC), Human thymus expressed, Human TNF-alpha, Human TNF-alpha, Human TNF-beta (LT-alpha), Human type CC chemokine eotaxin 3 protein sequence, Human type II interleukin-1 receptor, Human wild-type interleukin-4 (hIL-4) protein, Human ZCHEMO-8 protein, Humanized Anti-VEGF Antibodies, and fragments thereof, Humanized Anti-VEGF Antibodies, and fragments thereof, Hyaluronidase, ICE 10 kD subunit., ICE 20 kD subunit., ICE 22 kD subunit., Iduronate-2-sulfatase, Iduronidase, IL-1 alpha, IL-1 beta, IL-1 inhibitor (IL-10., IL-1 mature, IL-10 receptor, IL-11, IL-11, IL-12 p40 subunit., IL-13, IL-14, IL-15, IL-15 receptor, IL-17, IL-17 receptor, II-17 receptor, II-17 receptor, IL-19, IL-1i fragments, IL1-receptor antagonist, IL-21 (TIF), IL-3 containing fusion protein., IL-3 mutant proteins, IL-3 variants, IL-3 variants, IL-4, IL-4 mutein, IL-4 mutein Y124G, IL-4 mutein Y124X, IL-4 muteins, II-5 receptor, IL-6, II-6 receptor, IL-7 receptor clone, IL-8 receptor, IL-9 mature protein variant (Met117 version), immunoglobulins or immunoglobulin-based molecules or fragment of either (e.g. a Small Modular ImmunoPharmaceutical.TM. ("SMIP") or dAb, Fab' fragments, F(ab')2, scAb, scFv or scFv fragment), including but not limited to plasminogen, Influenza Vaccine, Inhibin alpha, Inhibin beta, insulin, insulin-like growth factor, Integrin Mab, inter-alpha trypsin inhibitor, inter-alpha trypsin inhibitor, Interferon gamma-inducible protein (IP-10), interferons (such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species), interferons (such as interferon alpha species and sub-species, interferon beta species and sub-species, interferon gamma species and sub-species), Interleukin 6, Interleukin 8 (IL-8) receptor, Interleukin 8 receptor B, Interleukin-1alpha, Interleukin-2 receptor associated protein p43, interleukin-3, interleukin-4 muteins, Interleukin-8 (IL-8) protein., interleukin-9, Interleukin-9 (IL-9) mature protein (Thr117 version), interleukins (such as IL10, IL11 and IL2), interleukins (such as IL10, IL11 and IL2), Japanese encephalitis vaccine, Kalikrein Inhibitor, Keratinocyte growth factor, Kunitz domain protein (such as aprotinin, amyloid precursor protein and those described in WO 03/066824, with or without albumin fusions), Kunitz domain protein (such as aprotinin, amyloid precursor protein and those described in WO 03/066824, with or without albumin fusions), LACI, lactoferrin, Latent TGF-beta binding protein II, leptin, Liver expressed chemokine-1 (LVEC-1), Liver expressed chemokine-2 (LVEC-2), LT-alpha, LT-beta, Luteinization Hormone, Lyme Vaccine, Lymphotactin, Macrophage derived chemokine analogue MDC (n+1), Macrophage derived chemokine analogue MDC-eyfy, Macrophage derived chemokine analogue MDC-yl, Macrophage derived chemokine, MDC, Macrophage-derived chemokine (MDC), Maspin; Protease Inhibitor 5, MCP-1 receptor, MCP-1a, MCP-1b, MCP-3, MCP-4 receptor, M-CSF, Melanoma inhibiting protein, Membrane-bound proteins, Met117 human interleukin 9, MIP-3 alpha, MIP-3 beta, MIP-Gamma, MIRAP, Modified Rantes, monoclonal antibody, MP52, Mutant Interleukin 6 S176R, myofibrillar contractile protein Troponin I, Natriuretic Peptide, Nerve Growth Factor-beta, Nerve Growth Factor-beta2, Neuropilin-1, Neuropilin-2, Neurotactin, Neurotrophin-3, Neurotrophin-4, Neurotrophin-4a, Neurotrophin-4b, Neurotrophin-4c, Neurotrophin-4d, Neutrophil activating peptide-2 (NAP-2), NOGO-66 Receptor, NOGO-A, NOGO-B, NOGO-C, Novel beta-chemokine designated PTEC, N-terminal modified chemokine GroHEK/hSDF-1alpha, N-terminal modified chemokine GroHEK/hSDF-1 beta., N-terminal modified chemokine met-hSDF-1 alpha, N-terminal modified chemokine met-hSDF-1 beta, OPGL, Osteogenic Protein-1; OP-1; BMP-7, Osteogenic Protein-2, OX40; ACT-4, OX40L, Oxytocin (Neurophysin I), parathyroid hormone, Patched, Patched-2, PDGF-D, Pertussis toxoid, Pituitary expressed chemokine (PGEC), Placental Growth Factor, Placental Growth Factor-2, Plasminogen Activator Inhibitor-1; PAI-1, Plasminogen Activator Inhibitor-2; PAI-2, Plasminogen Activator Inhibitor-2; PAI-2, Platelet derived growth factor, Platelet derived growth factor Bv-sis, Platelet derived growth factor precursor A, Platelet derived growth factor precursor B, Platelet Mab, platelet-derived endothelial cell growth factor (PD-ECGF), Platelet-Derived Growth Factor A chain, Platelet-Derived Growth Factor B chain, polypeptide used to treat sepsis, Preproapolipoprotein "milano" variant, Preproapolipoprotein "paris" variant, pre-thrombin, Primate CC chemokine "ILINCK", Primate CXC chemokine "IBICK", proinsulin, Prolactin, Prolactin2, prosaptide, Protease inhibitor peptides, Protein C, Protein S, pro-thrombin, prourokinase, RANTES, RANTES 8-68, RANTES 9-68, RANTES peptide, RANTES receptor, Recombinant interleukin-16, Resistin, restrictocin, Retroviral protease inhibitors, ricin, Rotavirus Vaccine, RSV Mab, saporin, sarcin, Secreted and Transmembrane polypeptides, Secreted and Transmembrane polypeptides, serum cholinesterase, serum protein (such as a blood clotting factor), Soluble BMP Receptor Kinase Protein-3, Soluble VEGF Receptor, Stem Cell Inhibitory Factor, Straphylococcus Vaccine, Stromal Derived Factor-1 alpha, Stromal Derived Factor-1 beta, Substance P (tachykinin), T1249 peptide, T20 peptide, T4 Endonuclease, TACI, Tarc, TGF-beta 1, TGF-beta 2, Thr117 human interleukin 9, thrombin, Thrombopoietin derivativel, Thrombopoietin derivative2, Thrombopoietin derivative3, Thrombopoietin derivative4, Thrombopoietin derivative5, Thrombopoietin derivative6, Thrombopoietin derivative7, Thymus expressed chemokine (TECK), Thyroid stimulating Hormone, tick anticoagulant peptide, Tim-1 protein, TNF-alpha precursor, TNF-R, TNF-RII; TNF p75 Receptor; Death Receptor, tPA, transferrin, transforming growth factor beta, Troponin peptides, Truncated monocyte chemotactic protein 2 (6-76), Truncated monocyte chemotactic protein 2 (6-76), Truncated RANTES protein (3-68), tumour necrosis factor, Urate Oxidase, urokinase, Vasopressin (Neurophysin II), VEGF R-3; flt-4, VEGF Receptor; KDR; flk-1, VEGF-110, VEGF-121, VEGF-138, VEGF-145, VEGF-162, VEGF-165, VEGF-182, VEGF-189, VEGF-206, VEGF-D, VEGF-E; VEGF-X, von Willebrand's factor, Wild type monocyte chemotactic protein 2, Wild type monocyte chemotactic protein 2, ZTGF-beta 9.

Chemotherapy Drugs

[0332] 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, A, Abraxane, Accutane.RTM., Actinomycin-D, Adriamycin.RTM., Adrucil.RTM., Agrylin.RTM., Ala-Cort.RTM., Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ.RTM., Alkeran.RTM., All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron.RTM., Anastrozole, Arabinosylcytosine, Ara-C, Aranesp.RTM., Aredia.RTM., Arimidex.RTM., Aromasin.RTM., Arranon.RTM., Arsenic Trioxide, Asparaginase, ATRA, Avastin.RTM., Azacitidine, BCG, BCNU, Bevacizumab, Bexarotene, BEXXAR.RTM., Bicalutamide, BiCNU, Blenoxane.RTM., Bleomycin, Bortezomib, Busulfan, Busulfex.RTM., C225 , Calcium Leucovorin, Campeth.RTM., Camptosar.RTM., Camptothecin-11, Capecitabine, Carac.TM., Carboplatin, Carmustine, Carmustine Wafer, Casodex.RTM., CC-5013, CCNU, CDDP, CeeNU, Cerubidine.RTM., Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen.RTM., CPT-11, Cyclophosphamide, Cytadren.RTM., Cytarabine, Cytarabine Liposomal, Cytosar-U.RTM., Cytoxan.RTM., Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome.RTM., Decadron, Decitabine, Delta-Cortef.RTM., Deltasone.RTM., Denileukin diftitox, DepoCyt.TM., Dexamethasone, Dexamethasone acetate , Dexamethasone Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil.RTM., Doxorubicin, Doxorubicin liposomal, Droxia.TM., DTIC, DTIC-Dome.RTM., Duralone.RTM., Efudex.RTM., Eligard.TM., Ellence.TM., Eloxatin.TM., Elspar.RTM., Emcyt.RTM., Epirubicin, Epoetin alfa, Erbitux.TM., Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos.RTM., Etoposide, Etoposide Phosphate, Eulexin.RTM., Evista.RTM., Exemestane, Fareston.RTM., Faslodex.RTM., Femara.RTM., Filgrastim, Floxuridine, Fludara.RTM., Fludarabine, Fluoroplex.RTM., Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR.RTM., Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar.RTM., Gleevec.TM., Gliadel.RTM. Wafer, GM-CSF, Goserelin, Granulocyte-Colony Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin.RTM., Herceptin.RTM., Hexadrol, Hexalen.RTM., Hexamethylmelamine, HMM, Hycamtin.RTM., Hydrea.RTM., Hydrocort Acetate.RTM., Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin.RTM., Idarubicin, Ifex.RTM., IFN-alpha , Ifosfamide, IL-11 , IL-2 , Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A.RTM. (interferon alfa-2b), Iressa.RTM., Irinotecan, Isotretinoin, Kidrolase.RTM., Lanacort.RTM., Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, Leukine.TM., Leuprolide, Leurocristine, Leustatin.TM., Liposomal Ara-C, Liquid Pred.RTM., Lomustine, L-PAM, L-Sarcolysin, Lupron.RTM., Lupron Depot.RTM., M, Matulane.RTM., Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone.RTM., Medrol.RTM., Megace.RTM., Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex.TM., Methotrexate, Methotrexate Sodium, Methylprednisolone, Meticorten.RTM., Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol.RTM., MTC, MTX, Mustargen.RTM., Mustine , Mutamycin.RTM., Myleran.RTM., Mylocel.TM., Mylotarg.RTM., Navelbine.RTM., Nelarabine, Neosar.RTM., Neulasta.TM., Neumega.RTM., Neupogen.RTM., Nexavar.RTM., Nilandron.RTM., Nilutamide, Nipent.RTM., Nitrogen Mustard, Novaldex.RTM., Novantrone.RTM., Octreotide, Octreotide acetate, Oncospar.RTM., Oncovin.RTM., Ontak.RTM., Onxal.TM., Oprevelkin, Orapred.RTM., Orasone.RTM., Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin.RTM., Paraplatin.RTM., Pediapred.RTM., PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON.TM., PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol.RTM., Platinol-AQ.RTM., Prednisolone, Prednisone, Prelone.RTM., Procarbazine, PROCRIT.RTM., Proleukin.RTM., Prolifeprospan 20 with Carmustine Implant, Purinethol.RTM., R, Raloxifene, Revlimid.RTM., Rheumatrex.RTM., Rituxan.RTM., Rituximab, Roferon-A.RTM. (Interferon Alfa-2a), Rubex.RTM., Rubidomycin hydrochloride, Sandostatin.RTM., Sandostatin LAR.RTM., Sargramostim, Solu-Cortef.RTM., Solu-Medrol.RTM., Sorafenib, SPRYCEL.TM., STI-571, Streptozocin, SU11248, Sunitinib, Sutent.RTM., Tamoxifen, Tarceva.RTM., Targretin.RTM., Taxol.RTM., Taxotere.RTM., Temodar.RTM., Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid.RTM., TheraCys.RTM., Thioguanine, Thioguanine Tabloid.RTM., Thiophosphoamide, Thioplex.RTM., Thiotepa, TICE.RTM., Toposar.RTM., Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, Trexall.TM., Trisenox.RTM., TSPA, TYKERB.RTM., VCR, Vectibix.TM., Velban.RTM., Velcade.RTM., VePesid.RTM., Vesanoid.RTM., Viadur.TM., Vidaza.RTM., Vinblastine, Vinblastine Sulfate, Vincasar Pfs.RTM., Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon.RTM., Xeloda.RTM., Zanosar.RTM., Zevalin.TM., Zinecard.RTM., Zoladex.RTM., Zoledronic acid, Zolinza, Zometa.RTM..

Radiopharmaceuticals

[0333] Carbon-11, Carbon-14, Chromium-51, Cobalt-57, Cobalt-58, Erbium-169, Fluorine-18, Gallium-67, Gold-198, Indium-111, Indium-113m, Iodine-123, Iodine-125, Iodine-131, Iron-59, Krypton-81m, Nitrogen-13, Oxygen-15, Phosphorous-32, Rhenium-186, Rubidium-82, Samarium-153, Selenium-75, Strontium-89, Technetium-99m, Thallium-201, Tritium, Xenon-127, Xenon-133, Yttrium-90.

Imaging Agents

[0334] Gadolinium, magnetite, manganese, technetium, I125, I131, P32, TI201, Iopamidol, PET-FDG.

Purification Tags

[0335] The albumin may also be fused to one or more purification tags such as (Ala-Trp-Trp-Pro).sub.n, avidin/streptavidin/Strep-tag, BCCP, B-tag (VP7 protein region of bluetongue virus), calmodulin binding protein (CBP), cellulose binding domains (CBD's), chitin binding domain, chloramphenicol acetyltransferase, c-myc, dihydrofolate reductase (DHFR), FLAG.TM. peptide (DYKDDDDK), galactose-binding protein, glutathione-S-transferase (GST), green flourescent protein (GFP), Growth hormone, N-terminus, hemagglutinin influenza virus (HAI), His-patch thioredoxin, His-tag, HSB-tag, KSI, lacZ (.beta.-Galactosidase), maltose binding protein (MBP), NusA, ompT/ompA/pelB/DsbA/DsbC, polyarginine, polyaspartic acid, polycysteine, polyphenyalanine, S-tag, staphylococcal protein A, streptococcal protein G, T4 gp55, T7gene10, T7-tag, thioredoxin, trpE, ubiquitin.

Ligand Binding

[0336] HSA has ligand binding and esterase activities, as described in "All about Albumin", T. Peters Jr., Academic Press N.Y. The ligand binding properties include binding to anionic and neutral ligands such as long-chain fatty acids, bilirubin and other miscellaneous ligands. The long-chain fatty acids, oleic (C18:1), palmitic (C16:0), linoleic (C18:2), stearic (C18:0), arachidonic (C20:4) and palmitoleic (C16:1) are known to bind HSA.

[0337] The polypeptide may include insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially reduce the useful ligand-binding, immunological or receptor binding properties of albumin, for example to FcRN, bilirubin and/or a fatty acid. The polypeptide may have at least 5%, 10%, 15%, 20%, 30%, 40% or 50%, 60%, 70%, at least 80%, 90%, 95%, 100%, 105% or more of human serum albumin's receptor binding activity, mole for mole. The polypeptide may have increased affinity for an albumin receptor.

[0338] Ligand binding studies can be performed on HSA and thio-albumins using an isothermal titration calorimetry method that had been suitably qualified for this purpose. Samples can be pre-treated by defatting (Sogami, M. and J. F. Foster (1968). Biochemistry 7(6): 2172-82, incorporated herein by reference) followed by thiol blocking (Sogami, M., H. A. Petersen, et al. (1969). Biochemistry 8(1): 49-58, incorporated herein by reference) and subsequent gel permeation chromatography. The binding curves generated for thio-albumins and HSA with octanoate, for example, may subsequently be compared, and functional similarity established.

Conjugation Methods

[0339] The albumin mutein (thio-albumin) of the invention can be covalently linked to one or more conjugation partners such as bioactive compounds by methods known in the art (for example those provided by Pierce, Thermo Fisher Scientific, Rockford, IL, USA; http://www.piercenet.com/files/1601361Crosslink.pdf). These include, but are not limited to incorporating or engineering a thiol reactive group into or onto the conjugation partner, for example by incorporating or engineering another free thiol present on the conjugation partner; or by incorporating or engineering a pyridyl disulphide group on the conjugation partner; or by incorporating or engineering an iodoacetyl group on the bioactive compound or or by incorporating or engineering a maleimide group on the conjugation partner. For example, N-ethylmaleimide (NEM, Pierce), 2-amino-2'-aminoethanethiolsulfonate (Pierce), N-beta-maleimidoprpionic acid (BMPA Pierce), methyl methane thiosulfonate (MMTS, Pierce), fluorescein-5-maleimide (Pierce), 5-iodoacetamido-fluorescein (5-IAF, Pierce) or N-[6-7-amino-4-methylcoumarin-3-acetamido) hexyl]-3'[2'-pyridyldithio] propionamide (AMCA-HPDP, Pierce).

[0340] If the conjugation partner contains at least one thiol group, then the conjugation partner may be cross-linked to the albumin mutein of the invention by methods known to the art such as, but not limited to, oxidation or by the use of cross-linking reagents such as, but not limited to, 1,4-Bis-maleimidibutane (BMB, Pierce); 1,4-Bis-maleimidyl-2,3-dihydroxybutane (BMDB, Pierce); Bis-maleimidohexane (BMH, Pierce), Bis-maleimidoethane (BMOE, Pierce); 1,8-Bis-Maleimidotriethyleneglycol (BM[PEO]3 Pierce); 1,11-Bis-Maleimidotetraethyleneglycol (BM[PEO]4 Pierce); 1,4-Di-[3'-(2'-pyridyldithio)-propionamido]butane (DPDPB, Pierce); dithuio-bis-maleimidoethane (DTME Pierce); 1,6-Hexane-bis-vinylsulfone (HBVS, Pierce) and Tris-[2-maleimimidoethyl]amine (TMEA, Pierce).

[0341] If the conjugation partner does not contain a thiol reactive group then it may be modified to incorporate one or more such groups by either chemical modification or genetic engineering by methods know to the art (Chapman, A.P. (2002) Adv. Drug Deliv. Rev., 54 531-545: Humphreys, D. P. et al. Protein Engineering, Design & Selection vol. 20 no. 5 pp. 227-234, 2007). While these two references describe methodologies to cross-link PEG to an engineered free thiol within an antibody or antibody fragment, the techniques may be used to cross-link a conjugation partner to an engineered free thiol within the albumin mutein of the invention. Alternatively the Drug Affinity Complex (DAC.TM.) technology developed by ConjuChem Inc. (Montreal, Quebec, Canada, H2X 3Y8) may be used, e.g. as described in WO200069902. There are three parts of each DAC.TM. construct: 1) the drug component (the portion responsible for biologic activity); 2) a linker attached to the drug component, and 3) a reactive chemistry group at the opposite end of the linker, usually a soft electrophile selective for thiols; a maleimide is the most useful embodiment. Other applicable conjugation methods are described in WO2007/071068 incorporated herein by reference.

[0342] If the conjugation partner does not contain a thiol reactive group but does contain one or more amino groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-5-azido-2-nitrobenzoyloxysuccinimide (AMAS, Pierce), N-[beta-maleimidopropyloxy] succinimide ester (BMPS, Pierce), N-eta-maleimidocaproic acid (EMCA, Pierce), N-[eta-maleimidocaproyloxy]succinimide ester (EMCS, Pierce), N-[eta-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS, Pierce), N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS, Pierce), N-[gamma-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS, Pierce), N-kappa-maleimidoundecanoic acid (KMUA, Pierce), N-[kappa -maleimidoundecanoic acid]hydrazide (KMUH, Pierce), N-[kappa -maleimidoundecanoyloxy]sulfosuccinimide ester (sulfo-KMUS, Pierce), m-maleimidobenzoyl-N-hydroxysuccinimide (MBS, Pierce), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS, Pierce), N-succinimidyl S-acetylthio-acetate (SATA, Pierce), N-succinimidyl S-acetylthiopropionate (SATP, Pierce), succinimidyl 3-[bromoacetamido]propionate (SBAP, Pierce), N-succinimidyl iodoacetate (SIA, Pierce), N-succinimidyl[4-iodoacetyl]aminobenzoate (STAB, Pierce), sulfosuccinimidyl[4-iodoacetyl]aminobenzoate (sulfo-SIAB, Pierce), succinimidyl [4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC, Pierce), sulfosuccinimidyl [4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC, Pierce), succinimidyl-[4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate (LC-SMCC, Pierce), 4-succininimidyloxycarbonyl-methyl-alpha[2-pyridyldithio]toluene (SMPT, Pierce), sulfosuccinimidyl-6-[alpha-methyl-alpha.quadrature.2-pyridyldith- io)toluamido]hexanoate (sulfo-LC-SMPT, Pierce), succinimidyl 4-[p-maleimidophenyl]-butyrate (SMPB, Pierce), sulfosuccinimidyl 4-[p-maleimidophenyl]-butyrate (sulfo-SMPB, Pierce), succinimidyl-6-[(beta-maleimidopropionamido)hexanoate] (SMPH, Pierce), N-succinimidyl 3-[2-pyridyldithio]propionate (SPDP, Pierce), succinimidyl [3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP, Pierce), sulfosuccinimidyl [3'-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP, Pierce) and N-succinimidyl-[4-vinylsulfonyl]benzoate (SVSB Pierce). It may be advantageous to block certain amine residue as described by Kavimandan et al., (2006) Bioconjugate Chem. 17, 1376-1384.

[0343] If the conjugation partner does not contain a thiol reactive group but does contain one or more carbonyl (oxidised carbohydrate) groups then it can be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[eta-maleimidocaproic acid]hydrazide (EMCH, Pierce), 4-[N-maleimidomethyl]cyclohexane-1carboxylhydrazide.HCl.1/2 dioxane (M2C2H, Pierce), 3-maleimidophenyl boronic acid (MPBH, Pierce) and 3-[2-pyridyldithio]propionyl hydrazide (PDPH, Pierce).

[0344] If the conjugation partner does not contain a thiol reactive group but does contain one or more hydroxyl groups then it may be modified to incorporate one or more thiol reactive groups by chemical modification by methods known to the art such as the use of cross-linking reagents such as, but not limited to, N-[p-maleimidophenyl]isocyanate (PMPI, Pierce).

Conjugation Competence of Albumin Variant

[0345] The conjugation competence of polypeptides of the invention may be tested by fluorescent labelling and cellular uptake, as described by McGraw et al., (1987), The Journal of Cell Biology, 105, 207-214 and Presley et al., (1993), The Journal of Cell Biology, 122, 1231-1241. Other methods of testing conjugation competence include conjugating the albumin to another molecule such as HRP. Subsequently, the mass of the resultant conjugate and/or the activity of the conjugated compound may be assayed, for example by mass spectrometry or by enzyme assay.

Microorganism.

[0346] A host strain suitable for use in the present invention includes an hsp150-deficient version of DXY1, disclosed in S. M. Kerry-Williams et al. (1998) Yeast 14:161-169. WO 95/33833 teaches the skilled person how to prepare hsp150-deficient yeast. This host strain may be referred to as `Strain 1`.

[0347] All documents cited are incorporated by reference in their entirety.

[0348] The invention is described by way of example only with reference to the following examples:

EXAMPLES

Example 1

Construction of Albumin Mutein Expression Plasmids

[0349] The HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 0 073 646 and EP 0 286 424. Expression plasmids for albumin variants of this invention can be constructed in a similar way to pDB2244 described in WO 00/44772 or pDB2305 described in WO/2006/013859 for expression of human serum albumin from S. cerevisiae. Plasmid pDB2305 contains the HSA sequence codon-optimised for expression in S. cerevisiae. Alternative codon optimisation methods may be used for the particular host organism selected for thio-albumin production. Expression plasmids for albumin variants of this invention can also be constructed in a similar way to those described in WO 2005/061719 A1 for improved expression of human serum albumin from S. cerevisiae.

[0350] Thio-albumin muteins can be made following modification of plasmid pDB2244 (FIG. 7) or pDB2305 by site directed mutagenesis. Overlapping mutagenic oligonucleotide sequences can be used to modify the codon of the selected residue(s) to any DNA sequence which encodes a cysteine residue (TGT or TGC) using the procedures indicated by a commercially available kit (such as Stratagene's Quikchange.TM. Kit). Alternatively, synthetic DNA fragments can be manufactured containing the desired modifications to the polynucleotide sequence.

Construction of a Thio-Albumin Mutant Expression Plasmids

[0351] Subcloning plasmids which may be used to create plasmid pDB2244 (FIG. 7) are plasmid pDB2243 (FIG. 8) (described in WO 00/44772) and pSAC35 (described in EP 286424). Plasmids pDB2243 and pDB2244 contain the native HSA gene. A skilled person will appreciate that the expression cassette may or may not be codon optimised; methods for constructing expression plasmids containing HSA codon optimised for expression in S. cerevisiae are described in WO/2006/013859. The native nucleotide sequence encoding HSA is provided in SEQ ID No. 2. A HSA nucleotide sequence codon-optimised for expression in S. cerevisiae is provided as SEQ ID No. 3.

[0352] Plasmid pDB2243 (6.203 kb) was digested to completion using restriction endonucleases Notl to release the 2.992 kb human serum albumin expression cassette.

[0353] Plasmid pSAC35 is derivative of pSAC3 by Chinery and Hinchliffe (1989) Curr. Genet. 16 , 21-25, and in EP 286424. Plasmid pSAC35 (11.037 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce 14.037 kb pDB2244 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene (FIG. 7). A person skilled in the art will appreciate that the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention.

[0354] Alternatively plasmid pDB2690 may be used. The construction of plasmid pDB2690 is described in WO/2005061719 A1. Plasmid pDB2690 (13.018 kb) was digested to completion with restriction endonuclease Notl and dephosphorylated using calf alkaline intestinal phosphatase and ligated with the 2.992 kb Notl human serum albumin expression cassette to produce a 16.039 kb plasmid pDB2713 which has the human serum albumin expression cassette orientated in the same direction as the LEU2 gene (FIG. 9). A person skilled in the art will appreciate that the expression cassette may or may not be codon optimised and that the expression cassette may or may not be cloned in either orientation in the expression vector as part of this invention.

[0355] As an alternative to site-directed mutagenesis expression plasmids for thio-albumin (i.e. conjugation competent albumin) variants of this invention could be made by subcloning synthesized DNA fragments into plasmid pDB2243 (FIG. 8) prior to cloning into pSAC35 or pDB2690. A method for the construction of a thio-albumin subcloning plasmid containing one extra conjugation competent cysteine (relative to SEQ ID No. 1) is described, by way of example only, below

[0356] The albumin DNA sequence of pDB2243 includes two Hindi II restriction endonuclease sites.

[0357] The synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected codon to a cysteine codon, or an existing cysteine codon is deleted or modified to a codon for another amino acid. Alternatively, the coding sequence for the mature thio-albumin may be extended at the 5' or 3' end(s) or insertions made within the polypeptide to add novel sequence(s) coding for cysteine or polypeptides containing one or more cysteine.

[0358] Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified at a selected cysteine codon to an alternative codon to create an unpaired cysteine. Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence is modified by substitution of two codons at a specified site to a cysteine codon (the amino acid chain length is reduced). Alternatively synthetic DNA may be modified such that the human serum albumin protein encoding sequence (e.g. SEQ ID No. 2 or SEQ ID No. 3 in relation to HSA) is modified by insertion of a cysteine codon at a specified site (the amino acid chain length is increased). Plasmid pDB2243 may be digested to completion with Hindlll restriction endonuclease and the fragment (approximately 4.383 kb) is recovered and dephosphorylated, the synthetic DNA containing the appropriate modification to the human serum albumin encoding sequence may then be cloned to produce the required thio-albumin subcloning plasmid. The thio-albumin subcloning plasmid may then be digested to produce an expression cassette, which may be cloned into a suitable expression plasmid in a similar manner to the construction of pDB2244, pDB2305 or pDB2713.

[0359] Those skilled in the art will appreciate that expression cassette for thio-albumin variants with additional modifications to the albumin protein sequence could be produced using a similar method to that described for the construction of a thio-albumin subcloning plasmid containing one extra conjugation competent cysteine (relative to SEQ ID No. 1).

[0360] A S. cerevisiae strain, e.g. Strain 1, may be transformed to leucine prototrophy with pDB2244 (WO 00/44772), or pDB2305 (WO/2006/013859) for expression of human serum albumin or the appropriate thio-albumin expression plasmids. Yeast may be transformed using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-1, protocol 2; Ito et al, 1983, J. Bacteriol., 153, 16; Elble, 1992, Biotechniques, 13, 18). Transformants may be selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates. The composition of BMMD is described by Sleep et al., 2002, Yeast, 18, 403. Cryopreserved stocks may be prepared in 20% (w/v) trehalose from 10 mL BMMD shake flask cultures (24 hours, 30.degree. C., 200 rpm).

Example 2

Expression of Albumin Muteins with Single Amino Acid Changes Compared to HSA

[0361] Thio-albumin variants with single amino acid changes were selected from Tables 5A, 5B and 6A. These variants were identified as the preferred mutations according to the methods described above. Details of each variant are given in FIG. 11, which provides a Construct Reference (e.g. TA1 for rHA A2C), the name of the plasmid encoding each thio-albumin variant expression construct and flanking sequences required for in vivo recombination by gap-repair, and the number given to a cryopreserved yeast stock (the yeast stock number) producing each thio-albumin variant. Details of the mutant codons compared to SEQ ID No. 2 are also provided, as are the SEQ ID numbers for each thio-albumin variant (DNA and protein).

[0362] To modify the amino acids in non-human serum albumins, the equivalent positions to a particular position in HSA may be determined from an alignment including human serum albumin (SEQ ID No. 1) such as FIGS. 2 and 3. The skilled person is familiar with alignments and can readily determine whether or not an amino acid in a sequence is equivalent to an amino acid in another sequence. For example, the position of the amino acid in the nonhuman albumin is not necessarily the same relative to the N-terminal end of HSA. For example, from FIG. 2 position 239 of HSA is an alanine residue, whereas the corresponding residue of the bovine sequence is serine-238. Similarly, valine-479 of HSA corresponds to leucine-478 of sheep albumin. The plasmid pDB3927 (FIG. 12) was constructed from plasmid pDB2244 (FIG. 7, WO 0044772A, `FL`: fusion leader sequence). pDB2244 was digested with restriction enzymes Swal and Hpal (both produce blunt ends) and self-ligated to form pDB3927. To create plasmid pDB3964 (FIG. 13) restriction enzyme sites were modified in the albumin DNA sequence (SEQ ID No. 2) of pDB3927 without modifying the protein sequence, as outlined below. The resultant DNA sequence is sequence ID No. 4.

[0363] 1) Introduced Enzyme Sites: (Basepair Positions in Brackets Refer to Positions in SEQ ID No. 4) SEQ ID No. 4

TABLE-US-00002 Restriction site SEQ ID No. SEQ ID No. 4 Position a SacII: GAGTCAGCTGAAAA .fwdarw.(to) GAGTCCGCGGAAAA (bp 173-178) b NheI/BmtI: AAGGCTTCGTCTGC .fwdarw.(to) AAGGCTAGCTCTGC (bp 571-576) c XhoI: TCTGCTTGAATGTGC .fwdarw.(to) TCTGCTCGAGTGTGC (bp 751-756) d BamHI: GTGGGCAGCAAAT .fwdarw.(to) GTGGGATCCAAAT (bp 751-756) e SalI: GGAAGTCGATGAAA .fwdarw.(to) GGAAGTCGACGAAA (bp 1477- 1482)

[0364] The coding sequence of HSA in pDB3964 is provided as SEQ ID No. 4. DNA synthesis and cloning was used to generate pDB3964 from pDB3927 (DNA2.0 Inc, USA). Synthetic DNA fragments were designed to alter specific amino acid codons within the albumin gene of pDB3964, or with combinations of modifications (see Example 3 below). DNA fragments containing these modifications were synthesised (DNA 2.0 Inc, USA) and cloned into pDB3964 to produce plasmids containing the thio-albumin sequences (FIG. 11). These synthetic genes and flanking regions were excised with restriction enzymes BstEII and BsrBI from the plasmids named in FIG. 11 for each of the thio-albumin variants and the controls pDB3927, pDB3964 and pDB2244, before purification of the resulting DNA fragments (PCR purification kit, Qiagen). The DNA fragments were used in the yeast transformation procedure described below to allow gap-repair in vivo with linearised pDB3936.

[0365] The plasmid pDB3853 (not shown) was constructed from base vector pDB2690 (Ref DB88/WO2005/061719A1) and the synthetic linker described below. The synthetic linker was constructed from two oligonucleotides (Sigma-Genosys) annealed in distilled water using a temperature gradient from 96.degree. C. to room temperature (1 min per 1.degree. C.). pDB2690 was digested using Kpnl and Notl, and purified by gel extraction (Qiagen), before ligation of the annealed linker:

TABLE-US-00003 `KpnI (linker) BamHI NotI` 5'- CGCTAGCCTCGAGGTTTAAACGCTAGCGAGCTCGGATCC -3' 3'- CATGGCGATCGGAGCTCCAAATTTGCGATCGCTCGAGCCTAGGCCG G -5'

[0366] Following the construction of pDB3853, the linker was excised using Pstl and Scal (3787bp fragment) before ligation into the gel extracted Pstl/Scal cut pSAC35 plasmid (WO 0044772A and WO2005/061719A1)), to form pDB3936 (FIG. 14).

[0367] pDB3936 was linearised with restriction enzymes Acc651 and BamHI before purification of the 9721bp fragment following separation by agarose gel electrophoresis. For the yeast transformation procedure to allow gap-repair in vivo (described below) the concentrations of the linearised pDB3936 and each of the BsrBI-BstEII fragments encoding the thio-albumin coding sequences was calculated and 100 ng of each use for each yeast transformation reaction.

[0368] Saccharomyces cerevisiae strain BXP10 was used as the expression host throughout (So-low, S. P., J. Sengbusch, et al. (2005). "Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae." Biotechnol Prog 21(2): 617-20.), although alternative expression hosts are also be suitable.

[0369] Cryopreserved stocks of S. cerevisiae BXP10 were prepared from 10 mL YEPPD (1% w/v yeast extract, 2% w/v plant peptone, 2% w/v dextrose)) shake flask cultures (grown for 24 hours, 30.degree. C., 200 rpm) mixed with an equal volume of 40% w/v sterile trehalose solution and dispensed in 1mL aliquots for storage at -80.degree. C. 10 mL BMMD, YEPPD and LB (1% w/v bacteriological tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl) shake flasks were inoculated with 100 .mu.L cryopreserved yeast stock and incubated for four days at 30.degree. C., 200 rpm as above before being observed microscopically to confirm they were axenic.

[0370] Frozen competent S. cerevisiae BXP10 cells were prepared by inoculating 100 .mu.L cryopreserved yeast stock into 10 mL YEPPD which were incubated for two days at 30.degree. C., 200 rpm, before being used to inoculate 300 mL YEPPD to an OD600=0.3. The cells were incubated as above for approximately 4 hours or until a doubling of OD.sub.600 had been achieved. The cells were harvested by centrifugation (3000.times.g, 5 min, room temperature) before resuspension in 120 mL distilled water followed by a further centrifugation step. The pellet was resuspended in 3 mL TE/LiAc (10 mM Tris, 1 mM EDTA, pH7; 500 mM lithium acetate) and glycerol added to a final concentration of 15% (v/v), before storage in aliquots at -80.degree. C.

[0371] S. cerevisiae BXP10 cells were transformed to leucine prototrophy using a modified lithium acetate method (Elble, R. "A simple and efficient procedure for transformation of yeasts." Biotechniques 13.1 (1992): 18-20. Ito, H., et al. "Transformation of intact yeast cells treated with alkali cations." J.Bacteriol. 153.1 (1983): 163-68.). 50 .mu.l of thawed competent cells were aliquoted into a 48-well microtitre plate (Nunc) before the addition of DNA fragments for gap-repair, as described above. The plate was mixed by swirling of the plate while flat on a benchtop. 300 .mu.l of PEG/LiAc (40% w/v PEG 3350, 100 mM lithium acetate) was added to each well and was mixed again. The plate was incubated at 30.degree. C. with shaking at 200 rpm for 1 hour before transfer to static incubation at 42.degree. C. for 30 min. After 1 min incubation on ice, the plate was centrifuged (2000.times.g, 5 min, room temperature) followed by removal of the supernatant and resuspension of the pellet in 200 .mu.l 1M sorbitol. The full volume was inoculated onto BMMD agar plates with CSM-Leu nutritional supplement (MP Biomedicals, Bio 101) and incubated for 4 days at 30.degree. C.

[0372] Single colony transformants were picked and patched onto fresh BMMD agar plates for short term storage. These patches were grown at 30.degree. C. and cells then inoculated into 10 mL BMMD shake flask cultures and cryopreserved as described earlier. 10 .mu.l of yeast stock was inoculated into a 48-well plate containing 0.5 mL BMMD per well. Growth of cultures in microtitre plates was achieved in a humidity chamber which was a sealed Perspex box containing wet paper towels to provide .about.100% humidity and evaporative loss below 0.25% over 5 days under growth conditions. The plates were incubated in the shaking humidity chamber (30.degree. C., 200 rpm,) for 5 days at 30.degree. C. The 48-well plate was centrifuged to pellet cells (2000.times.g, 10 min, room temperature) and the supernatant was harvested.

[0373] The concentration of the thio-albumin variants in the culture supernatants was determined by Gel Permeation High Pressure Liquid Chromatography (GP-HPLC). Protein concentrations were determined using a LC2010 HPLC system (Shimadzu) equipped with UV detection under Shimadzu VP7.3 client server software control. Injections of 25 .mu.L were made onto a 7.8 mm internal diameter.times.300 mm length TSK G3000SWXL column (Tosoh Bioscience), with a 6.0 mm internal diameter x 40 mm length TSK SW guard column (Tosoh Bioscience). Samples were chromatographed in 25 mM sodium phosphate, 100 mM sodium sulphate, 0.05% (w/v) sodium azide, pH 7.0 at 1 mL.min.sup.-1, with a run time of 15 minutes. Samples were quantified by UV detection at 280 nm, by peak height, relative to a recombinant human albumin standard of known concentration (10 mg/mL).

[0374] A non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of the thio-albumin variants with single mutations are compared against controls in FIG. 15. It is evident that all of the thio-albumin variants have been successfully secreted from S. cerevisiae BXP10. Preferred mutations have high expression titres and show a sharp Coomassie stained band equivalent to rHA controls by non-reducing SDS-PAGE analysis.

Example 3

Expression of Additional Thio-Albumin Variants

[0375] FIG. 16 describes an additional selection of thio-albumin variants with two or more free-thiol groups. Mutations shown to be expressed in Example 2 above were combined to generate sequences designed to have multiple free-thiol groups available for conjugation. This selection includes thio-albumin variants designed to have up to five free-thiol groups, thio-albumin variants designed to have free-thiol groups from within one Selection Group or from more than one Selection Group, thio-albumin variants designed to have free-thiol groups with and without the naturally occurring free-thiol at C34 of HSA, thio-albumin variants designed to have free-thiol groups from a range of Proximity Groups, and thio-albumin variants designed to have free-thiol groups derived from insertions, extensions, additions and/or deletions. It represents a sub-set of thio-albumins with multiple conjugation competent cysteine residues. The details of these thio-albumin variants, the plasmids encoding them and the SEQ ID No. for their DNA and protein sequences are described in FIG. 16, in a similar manner to those of FIG. 11 for the thio-albumin variants with single modifications. Methods for plasmid construction and expression from S. cerevisiae BXP10 are similar to those described above.

[0376] FIG. 17 shows a non-reducing SDS-PAGE analysis and the expression titres (by GP-HPLC) for each of these additional thio-albumin variants compared against an rHA control (pDB3927 coding sequence). Again, it is evident that all of the thio-albumin variants have been successfully secreted from S. cerevisiae BXP10. It therefore confirms that the selection criteria allow suitable thio-albumin variants to be generated and therefore indicates that there is no problem with undersirable mis-folding or aggregation. Again, preferred combinations of mutations have high expression titres and show a sharp Coomassie stained band equivalent to rHA controls by non-reducing SDS-PAGE analysis.

Example 4

Production, Purification and Conjugation of Thio-Albumin Variants

[0377] Five cryopreserved yeast stocks (9116, 9118, 9124, 9125 and 9130; FIG. 11) each in 1 mL aliquots were inoculated into shake flasks containing 100 mL BMMS growth medium (yeast nitrogen base without amino acids and (NH.sub.4).sub.2SO.sub.4, Difco 1.7 g/L; citric acid monohydrate 6.09 g/L; anhydrous Na.sub.2HPO.sub.4 20.16 g/L; (NH.sub.4).sub.2SO.sub.4 5.0 g/L; pH6.5.+-.0.2; sucrose added to 20 g/L). Cells were transferred from the shake flask to the fermenter (10 L working volume, Sartorius Biostat C 10-3 fermenter) when the concentration of cells in the shake flask has reached 0.8-1.2 g/L achieving a cell inocula concentration of .gtoreq.10 mg/L (greater than or equal to 10 mg/L) in the fermenter.

[0378] The thio-albumin variants proteins were produced by axenic culture of each of the five yeast strains in high cell density (HCD) fed-batch fermentation. The aim of the fermentation was to achieve maximum biomass and productivity by controlling feed rate addition so that formation of byproducts such as ethanol and acetate were avoided. Further details of the fermentation process are described in WO96/37515. The temperature and pH were controlled at 30.degree. C. and pH5.5 respectively. Culture supernatant was harvested by centrifugation using a Sorvall RC 3C centrifuge (DuPont) and frozen for storage, before being thawed for subsequent purification. Final product concentrations were determined by GP HPLC using a LC2010 HPLC system (Shimadzu) equipped with UV detection under Shimadzu VP7.3 client server software control as described above. FIG. 18 provides the yields of each thio-albumin variant (in g/L culture supernatant) and shows that high product titres of greater that 1 g/L culture supernatant were obtained in all cases.

[0379] A single step chromatography procedure was used to prepare material suitable for mass spectrometry. This purification step used a column (bed volume approximately 200 .mu.L) packed with AlbuPure.TM. matrix (ProMetic BioSciences Ltd, Cambridge UK or Novozymes Biophama UK Ltd.). This was equilibrated with 50 mM sodium phosphate, pH5.3, and loaded with neat culture supernatants, at approximately pH5.5-6.5, to approximately 40 mg protein/mL matrix. The column was washed with approximately 3 column volumes each of 50 mM sodium phosphate, pH5.3, and 50 mM ammonium acetate, pH8.0, respectively. Bound protein was eluted using approximately 5 column volumes of 50 mM ammonium acetate, 10 mM octanoate, pH7.0. The flow rate for the load step was 137 .mu.L/min, while the wash and elution steps were performed by means of centrifugal force, using a Heraeus Multifuge 3 centrifuge at 300 rpm. Final concentrations were in the range 1.8-4.0 mg/mL and samples were approximately 2 mL volume. Free thiol determination was performed immediately after sample elution by following the procedure described below.

[0380] The number of free thiols on a protein can be determined spectrophotometrically using Ellman's reagent. Ellman's reagent (5'5'-dithio-bis(2-nitronenzoic acid) (DTNB)) is an aromatic disulphide which reacts with thiol groups to form a mixed disulphide of the protein and one mole of 5-thio-2-nitrobenzoic acid (TNB) (per mole of protein sulfhydryl group). This reaction also results in a yellow colour from free TNB being released in solution. Alternatively the number of free thiols on a protein can be determined using mass spectrometric analysis of protein sample treated with DTNB reagent. 5-thio-2-nitrobenzoic acid (TNB) has a molecular weight of 199 Da, thus an increase in mass of 197 Da (TNB minus H.sub.2 lost during disulphide bond formation with the free thiol group on the test protein) indicates the presence of one free thiol group on the protein sample.

[0381] 700 .mu.L of the test protein sample was added to 100 .mu.l Buffer 2 (4 mg/mL DTNB and 500 mM Sodium Phosphate, pH 7.0) and 900 .mu.L Buffer 1 (0.1 M Tris-HCl, 100 mM EDTA, pH8.0). The preparation was allowed to mix for 25 minutes at ambient temperature (21-25.degree. C.) followed by filtration through a low molecular mass cut-off filter (Vivaspin 2-10000 MWCO Sartorius Stedim Germany). The filter was washed with two volumes of 0.1% Trifluoroacetic acid (TFA) and the sample was resuspended in 1 ml of 0.1% TFA. TNB labelled and unlabelled samples were prepared for mass spectrometric analysis by desalt-ing/concentrating using Solid Phase Extaction (SPE). SPE columns were prepared by first wetting with 1 mL of 70% Acetonitrile (ACN Fisher)/0.1% TFA and then equilibrating ready for loading with 0.1% TFA. 1 mL of sample was loaded on the equilibrated SPE columns allowing time for the protein to bind. The bound protein and SPE columns were then washed three times in 1 mL of 0.1% Formic acid (Merck). Finally the bound protein was eluted into pre-washed 1 mL microfuge tube with 0. 5mL 70% ACN/0.1%FA.

[0382] For Time-of-Flight mass spectrometry 30 .mu.L of sample was introduced into a hybrid quadru-pole time-of flight mass spectrometer (QqOaTOF, Applied Biosystems, QSTAR-XL.RTM.), equipped with an lonSpray.TM. source in positive ion mode, using flow injection analysis (FIA). The only instrument parameter that is actively tuned is the Decoupling Potential (DP), typically set to 250 V. Typically 2 minutes of sample scans are averaged. For protein analysis the TOF analyser is calibrated against protonated molecular ions of equine myoglobin (Sigma) and resolution is typically >14,000. Instrument control and data acquisition and processing were performed using Analyst.TM. QS v1.1 software (Applied Biosystems).

[0383] The results of the above analysis of the purified thio-albumin samples are described below. On addition of DTNB all samples quickly turned yellow as expected due to the presence of numerous free thiols. When the samples were visually compared to an equivalent sample of rHA, containing a single free thiol, the colour change observed for the thio-albumin samples was significantly more intense, strongly indicating the presence of multiple free thiols on each thio-albumin molecule. Results are summarised in FIG. 18, with increasing colour intensity increasing denoted by increased number of "+".

[0384] The thio-albumin variants produced at higher fermentation yields were preferred for analysis by the mass spectroscopy method described above. Therefore, the recombinant proteins rHA (A2C, L585C) (total of 3 free thiols), rHA (D129C, C360S, L585C) (total of 4 free thiols), and A2C rHA-Cys (total of 3 free thiols) were analysed by ESI TOF (electrospray ionsation time of flight) mass spectrometry pre- and post-DTNB treatment to determine the numbers of free thiols present on each molecule.

[0385] When rHA (A2C, L585C) was analysed pre-DTNB treatment (FIG. 19) the major deconvoluted peaks observed were at 66633 Da and 66807 Da which corresponds to 172 Da and 346 Da above the expected mass of 66461 Da. These modifications were likely to be due to species, of .about.172 Da present in the growth media cross linking to the free thiols in rHA (A2C, L585C). Post DTNB treatment mass spectrometric analysis (FIG. 20) resulted in a major a deconvoluted peak at 67428 Da which was 376 Da above the expected mass for the protein with 3 free thiols. This extra mass is most likely to be due to an extra TNB linked to a free thiol and a further 179 Da, this strongly suggests the presence of a 4 free thiols and a possible a further thiol blocked with a species of .about.179 Da. Hence, the rHA (A2C, L558C) thio-albumin variant is particularly surprising in that it provides more than the expected number of reactive groups available for conjugation. Also present is a series of peaks .about.396 Da apart which are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA (A2C, L558C) molecule. This adduct formation is known to occur in the presence of excess DTNB.

[0386] When rHA (D129C, C360S, L585C) (total of 4 free thiols) was analysed pre-DTNB treatment (FIG. 21) the major deconvoluted peaks observed were at 66575 Da and 66747 Da which corresponds to 172 Da and 344 Da above the expected mass of 66403 Da.

[0387] These modifications were likely to be due to molecules of .about.172 Da present in the growth media cross linking to the free thiols in rHA (D129C, C360S, L585C). Post DTNB treatment mass spectrometric analysis (FIG. 22) resulted in a major a deconvoluted peak at 67564 Da which was 373 Da above the expected mass for the protein with 4 free thiols. This extra mass is most likely to be due to an extra TNB linked to a free thiol and a further 176 Da, this strongly suggests the presence of a 5 free thiols and a possible a further thiol blocked with a species of .about.176 Da. Hence, the rHA (D129C, C360S, L585C) thio-albumin variant is particularly surprising in that it has provided more than the expected number of groups available for conjugation. Also present are a series of peaks .about.396 Da apart these are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA D129C, C360S, L585C. This adduct formation is known to occur in the presence of excess DTNB.

[0388] Finally when A2C rHA-Cys (total of 3 free thiols) was analysed pre-DTNB treatment (FIG. 23) the major deconvoluted peaks observed were at 66747 Da and 66919 Da which corresponds to 172 Da and 344 Da above the expected mass of 66574 Da. However also present was a smaller peak corresponding to the expected unmodified mass at 66575 Da. This mass spectra indicates the blocking of some free thiols while a proportion of the molecule is present containing the expected 3 free thiols. Post DTNB treatment mass spectrometric analysis (FIG. 24) resulted in a major a deconvoluted peak at 67142 Da which was 23 Da below the expected mass of 67165 Da of the protein labelled with 3 TNB molecules, this was likely due to the presence of two TNB molecules and a 175 Da modification, suggesting the presence of 3 thiols, one of which was blocked by the unknown .about.175 Da species. However on closer inspection of FIG. 24 the presence of secondary peaks 23 Da above each species can be seen. These secondary peaks correspond to small shoulders in the raw data (data not shown) which are likely to indicate the presence of the molecule modified with 3 TNB molecules, indicating 3 free thiols. The other major species present are due to excess DTNB still present at the time of ionisation causing DTNB adduct formation with the labelled rHA D129C, C360S, L585C. This adduct formation is known to occur in the presence of excess DTNB.

[0389] In conclusion, a range of thio-albumin variants have been produced with three or more conjugation competent cysteine residues. The conjugation competent cysteines can be in regions of that may or may not have secondary structure, and/or may or may not be generated from natural disulphide bonds, and/or may or may not be additional cysteines residues (such as cysteine residues extending from the natural C-terminus of HSA).

Example 5

Formation of Gels from Thio-Albumin Variants

[0390] Samples of TA35 (i.e. A2C, A364, D562 in addition to naturally occurring C34) and TA33 (i.e. A2C, L585C in addition to naturally occurring C34) were incubated at room-temperature for 24 hours and both formed gels.

Sequence CWU 1

1

1381585PRTHomo sapiensMISC_FEATURE(1)..(585)Human serum albumin (mature protein) 1Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 21758DNAArtificial SequencePolynucleotide coding sequence (not codon optimised) for human serum albumin 2gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 175831758DNAArtificial SequencePolynucleotide coding sequence (codon optimised for expression in yeast) for human serum albumin 3gacgctcaca agtccgaagt cgctcacaga ttcaaggact tgggtgaaga aaacttcaag 60gctttggtct tgatcgcttt cgctcaatac ttgcaacaat gtccattcga agatcacgtc 120aagttggtca acgaagttac cgaattcgct aagacttgtg ttgctgacga atctgctgaa 180aactgtgaca agtccttgca caccttgttc ggtgataagt tgtgtactgt tgctaccttg 240agagaaacct acggtgaaat ggctgactgt tgtgctaagc aagaaccaga aagaaacgaa 300tgtttcttgc aacacaagga cgacaaccca aacttgccaa gattggttag accagaagtt 360gacgtcatgt gtactgcttt ccacgacaac gaagaaacct tcttgaagaa gtacttgtac 420gaaattgcta gaagacaccc atacttctac gctccagaat tgttgttctt cgctaagaga 480tacaaggctg ctttcaccga atgttgtcaa gctgctgata aggctgcttg tttgttgcca 540aagttggatg aattgagaga cgaaggtaag gcttcttccg ctaagcaaag attgaagtgt 600gcttccttgc aaaagttcgg tgaaagagct ttcaaggctt gggctgtcgc tagattgtct 660caaagattcc caaaggctga attcgctgaa gtttctaagt tggttactga cttgactaag 720gttcacactg aatgttgtca cggtgacttg ttggaatgtg ctgatgacag agctgacttg 780gctaagtaca tctgtgaaaa ccaagactct atctcttcca agttgaagga atgttgtgaa 840aagccattgt tggaaaagtc tcactgtatt gctgaagttg aaaacgatga aatgccagct 900gacttgccat ctttggctgc tgacttcgtt gaatctaagg acgtttgtaa gaactacgct 960gaagctaagg acgtcttctt gggtatgttc ttgtacgaat acgctagaag acacccagac 1020tactccgttg tcttgttgtt gagattggct aagacctacg aaactacctt ggaaaagtgt 1080tgtgctgctg ctgacccaca cgaatgttac gctaaggttt tcgatgaatt caagccattg 1140gtcgaagaac cacaaaactt gatcaagcaa aactgtgaat tgttcgaaca attgggtgaa 1200tacaagttcc aaaacgcttt gttggttaga tacactaaga aggtcccaca agtctccacc 1260ccaactttgg ttgaagtctc tagaaacttg ggtaaggtcg gttctaagtg ttgtaagcac 1320ccagaagcta agagaatgcc atgtgctgaa gattacttgt ccgtcgtttt gaaccaattg 1380tgtgttttgc acgaaaagac cccagtctct gatagagtca ccaagtgttg tactgaatct 1440ttggttaaca gaagaccatg tttctctgct ttggaagtcg acgaaactta cgttccaaag 1500gaattcaacg ctgaaacttt caccttccac gctgatatct gtaccttgtc cgaaaaggaa 1560agacaaatta agaagcaaac tgctttggtt gaattggtca agcacaagcc aaaggctact 1620aaggaacaat tgaaggctgt catggatgat ttcgctgctt tcgttgaaaa gtgttgtaag 1680gctgatgata aggaaacttg tttcgctgaa gaaggtaaga agttggtcgc tgcttcccaa 1740gctgctttgg gtttgtaa 175841758DNAArtificial SequenceHA mutated to introduce restriction enzyme sites 4gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 175851758DNAArtificial sequenceTA1 HA with A2C 5gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 175861758DNAArtificial sequenceTA2 HA with D1C 6tgtgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 175871758DNAArtificial sequenceTA3 HA with C75S 7gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatctacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa

1740gctgccttag gcttataa 175881758DNAArtificial sequenceTA4 HA with T79C 8gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcatgtctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 175891758DNAArtificial sequenceTA5 HA with E82C 9gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgttgtacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758101758DNAArtificial sequenceTA6 HA with E86C 10gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggttgtat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758111758DNAArtificial sequenceTA7 HA with C124S 11gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt ctactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758121758DNAArtificial sequenceTA8 HA with C168S 12gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atcttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758131758DNAArtificial sequenceTA9 HA with C169S 13gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttctcaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758141758DNAArtificial sequenceTA10 HA with C91S 14gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tctgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758151758DNAArtificial sequenceTA11 HA with D121C 15gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360tgtgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758161758DNAArtificial sequenceTA12 HA with D129C 16gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt

240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcattgtaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758171758DNAArtificial sequenceTA13 HA with S270C 17gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattgt atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758181758DNAArtificial sequenceTA14HA with C316A 18gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgttgctaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758191758DNAArtificial sequenceTA16 HA with C360S 19gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtct 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758201758DNAArtificial sequenceTA17 HA with C361A 20gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080gctgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758211758DNAArtificial sequenceTA18 HA with C361S 21gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tctgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758221758DNAArtificial sequenceTA19 HA with A364C 22gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctt gtgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758231758DNAArtificial sequenceTA20 HA with Q397C 23gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagtg tcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758241758DNAArtificial sequenceTA21 HA with A504C 24gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg

cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatt gtgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758251758DNAArtificial sequenceTA22 HA with A578C 25gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc ttgtagtcaa 1740gctgccttag gcttataa 1758261758DNAArtificial sequenceTA23 HA with A581C 26gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740tgtgccttag gcttataa 1758271758DNAArtificial sequenceTA24 HA with C558S 27gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtcttgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758281758DNAArtificial sequenceTA25 HA with C567S 28gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctc ttttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758291758DNAArtificial sequenceTA26 HA with D549C 29gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatgtgtgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758301758DNAArtificial sequenceTA27 HA with D562C 30gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gcttgtgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758311758DNAArtificial sequenceTA28 HA with E505C 31gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg cttgtacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758321758DNAArtificial sequenceTA29 HA with L585C 32gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc

ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctgttaa 1758331758DNAArtificial sequenceTA33 HA with A2C and L585C 33gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctgttaa 1758341758DNAArtificial sequenceTA34 HA with A2C and A504C 34gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatt gtgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758351758DNAArtificial sequenceTA35 HA with A2C, A364C and D562C 35gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctt gtgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gcttgtgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758361758DNAArtificial sequenceTA36 HA with A2C, C34A, A364C and D562C 36gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagg ctccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctt gtgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gcttgtgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758371758DNAArtificial sequenceTA38 HA withA2C, A364C, D562C and L585C 37gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctt gtgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gcttgtgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctgttaa 1758381758DNAArtificial sequenceTA39 HA with C34A, A504C and E505C. 38gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagg ctccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatt gttgtacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758391758DNAArtificial sequenceTA41 HA with S270C and A581C 39gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattgt atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740tgtgccttag gcttataa 1758401758DNAArtificial sequenceTA43 HA with D129C, S270C and A581C 40gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcattgtaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattgt atctccagta aactgaagga atgctgtgaa

840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740tgtgccttag gcttataa 1758411758DNAArtificial sequenceTA46 HA with C169S 41gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttctcaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgttgctaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758421758DNAArtificial sequenceTA47 HA with D129C, C360S and L585C 42gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcattgtaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtct 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctgttaa 1758431761DNAArtificial sequenceTA51 HA with A2C and a cysteine immediately before the stop codon 43gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttatgtta a 1761441761DNAArtificial sequenceTA57 HA with A2C and a Cys insertion between G584 and L585 44gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gctgtttata a 1761451755DNAArtificial sequenceTA60 HA with A2C deltion of C316 45gattgtcaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgttaaaaa ctatgctgag 960gcaaaggatg tcttcctggg catgtttttg tatgaatatg caagaaggca tcctgattac 1020tctgtcgtgc tgctgctgag acttgccaag acatatgaaa ccactctaga gaagtgctgt 1080gccgctgctg atcctcatga atgctatgcc aaagtgttcg atgaatttaa acctcttgtg 1140gaagagcctc agaatttaat caaacaaaat tgtgagcttt ttgagcagct tggagagtac 1200aaattccaga atgcgctatt agttcgttac accaagaaag taccccaagt gtcaactcca 1260actcttgtag aggtctcaag aaacctagga aaagtgggat ccaaatgttg taaacatcct 1320gaagcaaaaa gaatgccctg tgcagaagac tatctatccg tggtcctgaa ccagttatgt 1380gtgttgcatg agaaaacgcc agtaagtgac agagtcacca aatgctgcac agaatccttg 1440gtgaacaggc gaccatgctt ttcagctctg gaagtcgacg aaacatacgt tcccaaagag 1500tttaatgctg aaacattcac cttccatgca gatatatgca cactttctga gaaggagaga 1560caaatcaaga aacaaactgc acttgttgag ctcgtgaaac acaagcccaa ggcaacaaaa 1620gagcaactga aagctgttat ggatgatttc gcagcttttg tagagaagtg ctgcaaggct 1680gacgataagg agacctgctt tgccgaggag ggtaaaaaac ttgttgctgc aagtcaagct 1740gccttaggct tataa 1755461758DNAArtificial sequenceTA63 HA with H39C and C253P 46gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agattgtgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagccag ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758471758DNAArtificial sequenceTA64 HA with C177F 47gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtccgcggaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctt tctgttgcca 540aagctcgatg aacttcggga tgaagggaag gctagctctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720gtccacacgg aatgctgcca tggagatctg ctcgagtgtg ctgatgacag ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080tgtgccgctg ctgatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gatccaaatg ttgtaaacat 1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg acgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 1758481767DNAArtificial sequenceTA65 HA with a Cys at the N-terminus and an Ala-Cys extension at the C-terminus 48tgtgatgcac acaagagtga ggttgctcat cggtttaaag atttgggaga agaaaatttc 60aaagccttgg tgttgattgc ctttgctcag tatcttcagc agtgtccatt tgaagatcat 120gtaaaattag tgaatgaagt aactgaattt gcaaaaacat gtgttgctga tgagtccgcg 180gaaaattgtg acaaatcact tcataccctt tttggagaca aattatgcac agttgcaact 240cttcgtgaaa cctatggtga aatggctgac tgctgtgcaa aacaagaacc tgagagaaat 300gaatgcttct tgcaacacaa agatgacaac ccaaacctcc cccgattggt gagaccagag 360gttgatgtga tgtgcactgc ttttcatgac aatgaagaga catttttgaa aaaatactta 420tatgaaattg ccagaagaca tccttacttt tatgccccgg aactcctttt ctttgctaaa 480aggtataaag ctgcttttac agaatgttgc caagctgctg ataaagctgc ctgcctgttg 540ccaaagctcg atgaacttcg ggatgaaggg aaggctagct ctgccaaaca gagactcaag 600tgtgccagtc tccaaaaatt tggagaaaga gctttcaaag catgggcagt agctcgcctg 660agccagagat ttcccaaagc tgagtttgca gaagtttcca agttagtgac agatcttacc 720aaagtccaca cggaatgctg ccatggagat ctgctcgagt gtgctgatga cagggcggac 780cttgccaagt atatctgtga aaatcaagat tcgatctcca gtaaactgaa ggaatgctgt 840gaaaaacctc tgttggaaaa atcccactgc attgccgaag tggaaaatga tgagatgcct 900gctgacttgc cttcattagc

tgctgatttt gttgaaagta aggatgtttg caaaaactat 960gctgaggcaa aggatgtctt cctgggcatg tttttgtatg aatatgcaag aaggcatcct 1020gattactctg tcgtgctgct gctgagactt gccaagacat atgaaaccac tctagagaag 1080tgctgtgccg ctgctgatcc tcatgaatgc tatgccaaag tgttcgatga atttaaacct 1140cttgtggaag agcctcagaa tttaatcaaa caaaattgtg agctttttga gcagcttgga 1200gagtacaaat tccagaatgc gctattagtt cgttacacca agaaagtacc ccaagtgtca 1260actccaactc ttgtagaggt ctcaagaaac ctaggaaaag tgggatccaa atgttgtaaa 1320catcctgaag caaaaagaat gccctgtgca gaagactatc tatccgtggt cctgaaccag 1380ttatgtgtgt tgcatgagaa aacgccagta agtgacagag tcaccaaatg ctgcacagaa 1440tccttggtga acaggcgacc atgcttttca gctctggaag tcgacgaaac atacgttccc 1500aaagagttta atgctgaaac attcaccttc catgcagata tatgcacact ttctgagaag 1560gagagacaaa tcaagaaaca aactgcactt gttgagctcg tgaaacacaa gcccaaggca 1620acaaaagagc aactgaaagc tgttatggat gatttcgcag cttttgtaga gaagtgctgc 1680aaggctgacg ataaggagac ctgctttgcc gaggagggta aaaaacttgt tgctgcaagt 1740caagctgcct taggcttagc ttgttaa 176749609PRTArtificial SequenceHA with fusion leader sequence 49Met Lys Trp Val Ser Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Ser Leu Asp Lys Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu 50585PRTArtificial sequenceTA1 HA with A2C 50Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 51585PRTArtificial sequenceTA2 HA with D1C 51Cys Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 52585PRTArtificial sequenceTA3 HA with C75S 52Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Ser Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys

Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 53585PRTArtificial sequenceTA4 HA with T79C 53Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Cys Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 54585PRTArtificial sequenceTA5 HA with E82C 54Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Cys Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 55585PRTArtificial sequenceTA6 HA with E86C 55Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Cys Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 56585PRTArtificial sequenceTA7 HA with C124S 56Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Ser Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys

Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 57585PRTArtificial sequenceTA8 HA with C168S 57Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Ser Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 58585PRTArtificial sequenceTA9 HA with C169S 58Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Ser Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 59585PRTArtificial sequenceTA10 HA with C91S 59Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Ser Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 60585PRTArtificial sequenceTA11 HA with D121C 60Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Cys Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245

250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 61585PRTArtificial sequenceTA12 HA with D129C 61Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Cys Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 62585PRTArtificial sequenceTA13 HA with S270C 62Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Cys Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 63585PRTArtificial sequenceTA14 HA with C316A 63Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Ala Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 64585PRTArtificial sequenceTA16 HA with C360S 64Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala

Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Ser Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 65585PRTArtificial sequenceTA17 HA with C361A 65Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 66585PRTArtificial sequenceTA18 HA with C361S 66Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Ser Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 67585PRTArtificial sequenceTA19 HA with A364C 67Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Cys Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 68585PRTArtificial sequenceTA20 HA with Q397C 68Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile

Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Cys Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 69585PRTArtificial sequenceTA21 HA with A504C 69Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Cys Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 70585PRTArtificial sequenceTA22 HA with A578C 70Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Cys Ser Gln Ala Ala Leu Gly Leu 580 585 71585PRTArtificial sequenceTA23 HA with A581C 71Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Cys Ala Leu Gly Leu 580 585 72585PRTArtificial sequenceTA24 HA with C558S 72Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70

75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Ser Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 73585PRTArtificial sequenceTA25 HA with C567S 73Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Ser Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 74585PRTArtificial sequenceTA26 HA with D549C 74Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Cys Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 75585PRTArtificial sequenceTA27 HA with D562C 75Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Cys Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 76585PRTArtificial sequenceTA28 HA with E505C 76Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val

Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Cys Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 77585PRTArtificial sequenceTA29 HA with L585C 77Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Cys 580 585 78585PRTArtificial sequenceTA33 HA with A2C and L585C 78Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Cys 580 585 79585PRTArtificial sequenceTA34 HA with A2C and A504C 79Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Cys Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val

Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 80585PRTArtificial sequenceTA35 HA with A2C, A364C and D562C 80Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Cys Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Cys Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 81585PRTArtificial sequenceTA36 HA with A2C, A364C and D562C 81Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ala Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Cys Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Cys Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 82585PRTArtificial sequenceTA38 HA with A2C, A364C, D562C and L585C 82Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Cys Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Cys Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Cys 580 585 83585PRTArtificial sequenceTA39 HA with C34A, A504C and E505C. 83Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Ala Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475

480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Cys Cys Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 84585PRTArtificial sequenceTA41 HA with S270C and A581C 84Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Cys Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Cys Ala Leu Gly Leu 580 585 85585PRTArtificial sequenceTA43 HA with D129C, S270C and A581C 85Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Cys Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Cys Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Cys Ala Leu Gly Leu 580 585 86585PRTArtificial sequenceTA46 HA with C169S 86Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Ser Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Ala Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 87585PRTArtificial sequenceTA47 HA with D129C and L585C 87Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Cys Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Ser Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410

415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Cys 580 585 88586PRTArtificial sequenceTA51 HA with A2C and a cysteine immediately before the stop codon 88Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Cys 580 585 89586PRTArtificial sequenceTA57 HA with A2C and an insertion between G584 and L585 89Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Cys Leu 580 585 90584PRTArtificial sequenceTA60 HA with A2C and deletion of C316 90Asp Cys His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Lys Asn Tyr Ala Glu 305 310 315 320 Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg 325 330 335 His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr 340 345 350 Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys 355 360 365 Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln 370 375 380 Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr 385 390 395 400 Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln 405 410 415 Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val 420 425 430 Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala 435 440 445 Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu 450 455 460 Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu 465 470 475 480 Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr 485 490 495 Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile 500 505 510 Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu 515 520 525 Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys 530 535 540 Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala 545 550 555 560 Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala 565 570 575 Ala Ser Gln Ala Ala Leu Gly Leu 580 91585PRTArtificial sequenceTA63 HA with H39C and C253P 91Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp Cys Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Pro Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu

Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 92585PRTArtificial sequenceTA64 HA with C177F 92Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Phe Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 93588PRTArtificial sequenceTA65 HA with a Cys at the N-terminus and an Ala-Cys extension at the C-terminus of HA 93Cys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly 1 5 10 15 Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu 20 25 30 Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr 35 40 45 Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp 50 55 60 Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr 65 70 75 80 Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu 85 90 95 Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn 100 105 110 Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe 115 120 125 His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala 130 135 140 Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys 145 150 155 160 Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala 165 170 175 Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala 180 185 190 Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly 195 200 205 Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe 210 215 220 Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr 225 230 235 240 Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp 245 250 255 Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile 260 265 270 Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser 275 280 285 His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro 290 295 300 Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr 305 310 315 320 Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala 325 330 335 Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys 340 345 350 Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His 355 360 365 Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu 370 375 380 Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 385 390 395 400 Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val 405 410 415 Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 420 425 430 Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 435 440 445 Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 450 455 460 His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 465 470 475 480 Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu 485 490 495 Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala 500 505 510 Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 515 520 525 Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 530 535 540 Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 545 550 555 560 Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu 565 570 575 Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Ala Cys 580 585 94607PRTBos taurus 94Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val 50 55 60 Lys Leu Val Asn Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser His Ala Gly Cys Glu Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Glu Leu Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala 100 105 110 Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser 115 120 125 His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn 130 135 140 Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys 145 150 155 160 Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170 175 Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys 180 185 190 Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Glu Thr Met 195 200 205 Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala 210 215 220 Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala 225 230 235 240 Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Val Glu Val Thr Lys 245 250 255 Leu Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285 Asp Asn Gln Asp Thr Ile Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys 290 295 300 Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Lys Asp Ala 305 310 315 320 Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys 325 330 335 Asp Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Ala Phe Leu Gly Ser 340 345 350 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val 355 360 365 Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys 370 375 380 Ala Lys Asp Asp Pro His Ala Cys Tyr Ser Thr Val Phe Asp Lys Leu 385 390 395 400 Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Asp 405 410 415 Gln Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val 420 425 430 Arg Tyr Thr Arg Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Val Ser Arg Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro 450 455 460 Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490 495 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Thr Pro Asp Glu Thr Tyr Val Pro Lys Ala Phe Asp Glu Lys 515 520 525 Leu Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys 530 535 540 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro 545 550 555 560 Lys Ala Thr Glu Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala 565 570 575 Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala 580 585 590 Val Glu Gly Pro Lys Leu Val Val Ser Thr Gln Thr Ala Leu Ala 595 600 605 95608PRTFelis catus 95Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Thr Arg Arg Glu Ala His Gln Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Leu Gly Glu Glu His Phe Arg Gly Leu Val Leu 35 40 45 Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Gly Cys Val Ala Asp 65 70 75 80 Gln Ser Ala Ala Asn Cys Glu Lys Ser Leu His Glu Leu Leu Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Ser Leu Arg Asp Lys Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Glu Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Gly Phe Gly Gln Leu Val Thr Pro Glu Ala 130 135 140 Asp Ala Met Cys Thr Ala Phe His Glu Asn Glu Gln Arg Phe Leu Gly 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Glu Tyr Lys Gly Val Phe Thr Glu Cys 180 185 190 Cys Glu Ala Ala Asp Lys Ala Ala Cys Leu Thr Pro Lys Val Asp Ala 195 200 205 Leu Arg Glu Lys Val Leu Ala Ser Ser Ala Lys Glu Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val 225 230 235 240 Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Ala Glu Ile Ser 245 250

255 Lys Leu Val Thr Asp Leu Ala Lys Ile His Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Thr Lys Leu Lys Glu Cys Cys Gly 290 295 300 Lys Pro Val Leu Glu Lys Ser His Cys Ile Ser Glu Val Glu Arg Asp 305 310 315 320 Glu Leu Pro Ala Asp Leu Pro Pro Leu Ala Val Asp Phe Val Glu Asp 325 330 335 Lys Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Thr Asp Asp Pro Pro Ala Cys Tyr Ala His Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro His Asn Leu Val Lys Thr Asn Cys 405 410 415 Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Thr His 450 455 460 Pro Glu Ala Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Gln Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Ser Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu 530 535 540 Lys Gln Ile Lys Lys Gln Ser Ala Leu Val Glu Leu Leu Lys His Lys 545 550 555 560 Pro Lys Ala Thr Glu Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly 565 570 575 Ser Phe Val Asp Lys Cys Cys Ala Ala Glu Asp Lys Glu Ala Cys Phe 580 585 590 Ala Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Ala 595 600 605 96608PRTCanis lupus familaris 96Met Lys Trp Val Thr Phe Ile Ser Leu Phe Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Leu Val Arg Arg Glu Ala Tyr Lys Ser Glu Ile Ala 20 25 30 His Arg Tyr Asn Asp Leu Gly Glu Glu His Phe Arg Gly Leu Val Leu 35 40 45 Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Ala Lys Glu Val Thr Glu Phe Ala Lys Ala Cys Ala Ala Glu 65 70 75 80 Glu Ser Gly Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Ser Leu Arg Asp Lys Tyr Gly Asp Met Ala 100 105 110 Asp Cys Cys Glu Lys Gln Glu Pro Asp Arg Asn Glu Cys Phe Leu Ala 115 120 125 His Lys Asp Asp Asn Pro Gly Phe Pro Pro Leu Val Ala Pro Glu Pro 130 135 140 Asp Ala Leu Cys Ala Ala Phe Gln Asp Asn Glu Gln Leu Phe Leu Gly 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Gln Gln Tyr Lys Gly Val Phe Ala Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Gly Pro Lys Ile Glu Ala 195 200 205 Leu Arg Glu Lys Val Leu Leu Ser Ser Ala Lys Glu Arg Phe Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ser Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Ala Glu Ile Ser 245 250 255 Lys Val Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp 290 295 300 Lys Pro Val Leu Glu Lys Ser Gln Cys Leu Ala Glu Val Glu Arg Asp 305 310 315 320 Glu Leu Pro Gly Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330 335 Lys Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Glu Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Thr Asp Asp Pro Pro Thr Cys Tyr Ala Lys Val Leu Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Asp Glu Pro Gln Asn Leu Val Lys Thr Asn Cys 405 410 415 Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Lys Leu Gly Lys Val Gly Thr Lys Cys Cys Lys Lys 450 455 460 Pro Glu Ser Glu Arg Met Ser Cys Ala Glu Asp Phe Leu Ser Val Val 465 470 475 480 Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg 485 490 495 Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Gly Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu 530 535 540 Lys Gln Val Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys 545 550 555 560 Pro Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly 565 570 575 Ala Phe Val Glu Lys Cys Cys Ala Ala Glu Asn Lys Glu Gly Cys Phe 580 585 590 Ser Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Val 595 600 605 97607PRTEquus asinus 97Met Lys Trp Val Thr Phe Val Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Phe Arg Gly Val Leu Arg Arg Asp Thr His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Leu Gly Glu Lys His Phe Lys Gly Leu Val Leu 35 40 45 Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Lys Cys Ala Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr 115 120 125 His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135 140 Ala Gln Cys Ala Ala Phe Gln Glu Asp Pro Asp Lys Phe Leu Gly Lys 145 150 155 160 Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly Pro Glu 165 170 175 Leu Leu Phe His Ala Glu Glu Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185 190 Pro Ala Asp Asp Lys Ala Gly Cys Leu Ile Pro Lys Leu Asp Ala Leu 195 200 205 Lys Glu Arg Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215 220 Ser Phe Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val Ala 225 230 235 240 Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser Lys 245 250 255 Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Thr Lys Tyr Ile Cys 275 280 285 Glu His Gln Asp Ser Ile Ser Gly Lys Leu Lys Ala Cys Cys Asp Lys 290 295 300 Pro Leu Leu Gln Lys Ser His Cys Ile Ala Glu Val Lys Glu Asp Asp 305 310 315 320 Leu Pro Ser Asp Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys 325 330 335 Glu Ile Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345 350 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360 365 Leu Leu Arg Ile Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys Cys Cys 370 375 380 Ala Glu Ala Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp Gln Phe 385 390 395 400 Thr Pro Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn Cys Asp 405 410 415 Leu Phe Glu Glu Val Gly Glu Tyr Asp Phe Gln Asn Ala Leu Ile Val 420 425 430 Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Ile Gly Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455 460 Glu Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Ile 485 490 495 Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Glu Leu Asp Glu Gly Tyr Ile Pro Lys Glu Phe Lys Ala Glu 515 520 525 Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Asp Glu Lys 530 535 540 Gln Ile Lys Lys Gln Ser Ala Leu Ala Glu Leu Val Lys His Lys Pro 545 550 555 560 Lys Ala Thr Lys Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala 565 570 575 Phe Val Ala Lys Cys Cys Gly Ala Glu Asp Lys Glu Ala Cys Phe Ala 580 585 590 Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala 595 600 605 98609PRTMeriones unguiculatus 98Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala 1 5 10 15 Phe Ser Arg Gly Val Phe Arg Arg Asp Ala Ala His Lys Ser Glu Ile 20 25 30 Ala His Arg Tyr Lys Asp Leu Gly Glu Lys Tyr Phe Lys Gly Leu Val 35 40 45 Leu Tyr Thr Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Glu Glu His 50 55 60 Val Lys Leu Val Arg Glu Val Thr Asp Phe Ala Ser Asn Cys Ala Lys 65 70 75 80 Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly 85 90 95 Asp Lys Leu Cys Ser Leu Pro Asn Phe Gly Glu Lys Tyr Ala Glu Met 100 105 110 Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu 115 120 125 Gln His Lys Asp Asp Asn Pro Gln Leu Pro Pro Phe Lys Arg Ala Glu 130 135 140 Pro Asp Ala Met Cys Thr Ala Phe Gln Glu Asn Ala Glu Ala Phe Met 145 150 155 160 Gly His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly 165 170 175 Pro Glu Leu Leu Tyr Leu Ala Asp Lys Tyr Thr Ala Val Leu Thr Glu 180 185 190 Cys Cys Ala Ala Asp Asp Lys Gly Ala Cys Leu Thr Pro Lys Leu Asp 195 200 205 Ala Leu Lys Glu Lys Ala Leu Val Ser Ala Val Arg Gln Arg Leu Lys 210 215 220 Cys Ser Ser Met Lys Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala 225 230 235 240 Val Ala Arg Met Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile 245 250 255 Thr Lys Leu Ala Thr Asp Leu Thr Lys Val Thr Gln Glu Cys Cys His 260 265 270 Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr 275 280 285 Met Cys Glu Asn Gln Ala Ser Ile Ser Ser Lys Leu Gln Ala Cys Cys 290 295 300 Asp Lys Glu Met Leu Gln Lys Ser Gln Cys Leu Ala Glu Val Glu His 305 310 315 320 Asp Asp Met Pro Ala Asp Leu Pro Ala Leu Thr Ala Asp Phe Val Glu 325 330 335 Asp Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu 340 345 350 Gly Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ser Val 355 360 365 Ser Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys 370 375 380 Cys Cys Ala Glu Ala Asp Pro His Ala Cys Tyr Gly His Val Phe Asp 385 390 395 400 Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Val Lys Ser Asn 405 410 415 Cys Glu Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Val 420 425 430 Leu Val Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu 435 440 445 Val Glu Ala Ala Arg Ser Leu Gly Arg Val Gly Thr His Cys Cys Ala 450 455 460 Leu Pro Glu Lys Lys Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala 465 470 475 480 Ile Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu 485 490 495 Gln Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys 500 505 510 Phe Ser Ala Leu Pro Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys 515 520 525 Ala Glu Thr Phe Thr Phe His Ala Asn Ile Cys Thr Leu Pro Glu Lys 530 535 540 Glu Lys Gln Met Glu Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His 545 550 555 560 Lys Pro Gln Ala Thr Glu Glu Gln Leu Lys Lys Val Met Gly Asp Phe 565 570 575 Ala Glu Phe Leu Glu Lys Cys Cys Lys Gln Glu Asp Lys Glu Ala Cys 580 585 590 Phe Ser Thr Glu Gly Pro Lys Leu Val Ala Glu Ser Gln Lys Ala Leu 595 600 605 Ala 99583PRTCapra hircus 99Asp Thr His Lys Ser Glu Ile Ala His Arg Phe Asn Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Gln Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Asp Glu His Val Lys Leu Val Lys Glu Leu Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser His Ala Gly Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Glu Leu Cys Lys Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Lys His Lys Asp Asp Ser Pro Asp Leu 100 105 110 Pro Lys Leu Lys Pro Glu Pro Asp Thr Leu Cys Ala Glu

Phe Lys Ala 115 120 125 Asp Glu Lys Lys Phe Trp Gly Lys Tyr Leu Tyr Glu Val Ala Arg Arg 130 135 140 His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Asn Lys Tyr 145 150 155 160 Asn Gly Val Phe Gln Glu Cys Cys Gln Ala Glu Asp Lys Gly Ala Cys 165 170 175 Leu Leu Pro Lys Ile Glu Thr Met Arg Glu Lys Val Leu Ala Ser Ser 180 185 190 Ala Arg Gln Arg Leu Arg Cys Ala Ser Ile Gln Lys Phe Gly Glu Arg 195 200 205 Ala Leu Lys Ala Trp Ser Val Ala Arg Leu Ser Gln Lys Phe Pro Lys 210 215 220 Ala Asp Phe Thr Asp Val Thr Lys Ile Val Thr Asp Leu Thr Lys Val 225 230 235 240 His Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg 245 250 255 Ala Asp Leu Ala Lys Tyr Ile Cys Asp His Gln Asp Thr Leu Ser Ser 260 265 270 Lys Leu Lys Glu Cys Cys Asp Lys Pro Val Leu Glu Lys Ser His Cys 275 280 285 Ile Ala Glu Ile Asp Lys Asp Ala Val Pro Glu Asn Leu Pro Pro Leu 290 295 300 Thr Ala Asp Phe Ala Glu Asp Lys Glu Val Cys Lys Asn Tyr Gln Glu 305 310 315 320 Ala Lys Asp Val Phe Leu Gly Ser Phe Leu Tyr Glu Tyr Ser Arg Arg 325 330 335 His Pro Glu Tyr Ala Val Ser Val Leu Leu Arg Leu Ala Lys Glu Tyr 340 345 350 Glu Ala Thr Leu Glu Asp Cys Cys Ala Lys Glu Asp Pro His Ala Cys 355 360 365 Tyr Ala Thr Val Phe Asp Lys Leu Lys His Leu Val Asp Glu Pro Gln 370 375 380 Asn Leu Ile Lys Lys Asn Cys Glu Leu Phe Glu Lys His Gly Glu Tyr 385 390 395 400 Gly Phe Gln Asn Ala Leu Ile Val Arg Tyr Thr Arg Lys Ala Pro Gln 405 410 415 Val Ser Thr Pro Thr Leu Val Glu Ile Ser Arg Ser Leu Gly Lys Val 420 425 430 Gly Thr Lys Cys Cys Ala Lys Pro Glu Ser Glu Arg Met Pro Cys Thr 435 440 445 Glu Asp Tyr Leu Ser Leu Ile Leu Asn Arg Leu Cys Val Leu His Glu 450 455 460 Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu Ser Leu 465 470 475 480 Val Asn Arg Arg Pro Cys Phe Ser Asp Leu Thr Leu Asp Glu Thr Tyr 485 490 495 Val Pro Lys Pro Phe Asp Gly Glu Ser Phe Thr Phe His Ala Asp Ile 500 505 510 Cys Thr Leu Pro Asp Thr Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu 515 520 525 Val Glu Leu Leu Lys His Lys Pro Lys Ala Thr Asp Glu Gln Leu Lys 530 535 540 Thr Val Met Glu Asn Phe Val Ala Phe Val Asp Lys Cys Cys Ala Ala 545 550 555 560 Asp Asp Lys Glu Gly Cys Phe Leu Leu Glu Gly Pro Lys Leu Val Ala 565 570 575 Ser Thr Gln Ala Ala Leu Ala 580 100608PRTCavia porcellus 100Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Val 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Leu Gly Glu Gly His Phe Lys Gly Leu Val Leu 35 40 45 Ile Thr Leu Ser Gln His Leu Gln Lys Ser Pro Phe Glu Glu His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Asp Phe Ala Lys Ala Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Gln Asn Cys Gly Lys Ala Ile Ala Thr Leu Phe Gly Asp 85 90 95 Lys Val Cys Ala Ile Pro Ser Leu Arg Glu Thr Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Ala Lys Glu Asp Pro Asp Arg Val Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Glu Arg Pro Glu Pro 130 135 140 Glu Ala Leu Cys Thr Ala Phe Lys Glu Asn Asn Asp Arg Phe Ile Gly 145 150 155 160 His Tyr Leu Tyr Glu Val Ser Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Lys Asn Ala Leu Thr Glu Cys 180 185 190 Cys Glu Ala Ala Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200 205 Ile Lys Glu Lys Ala Leu Val Ser Ser Ala Gln Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val 225 230 235 240 Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Ala Glu Ile Ser 245 250 255 Thr Ile Val Thr Ser Leu Thr Lys Val Thr Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Gln Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu His Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Val 290 295 300 Lys Pro Thr Leu Gln Lys Ala His Cys Ile Leu Glu Ile Gln Arg Asp 305 310 315 320 Glu Leu Pro Thr Glu Leu Pro Asp Leu Ala Val Asp Phe Val Glu Asp 325 330 335 Lys Glu Val Cys Lys Asn Phe Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ser Ile Gly 355 360 365 Met Leu Leu Arg Ile Ala Lys Gly Tyr Glu Ala Lys Leu Glu Lys Cys 370 375 380 Cys Ala Glu Ala Asp Pro His Ala Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Leu Gln Pro Leu Ile Asp Glu Pro Lys Lys Leu Val Gln Gln Asn Cys 405 410 415 Glu Leu Phe Asp Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ala 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Tyr Ala Arg Lys Leu Gly Ser Val Gly Thr Lys Cys Cys Ser Leu 450 455 460 Pro Glu Thr Glu Arg Leu Ser Cys Thr Glu Asn Tyr Leu Ala Leu Ile 465 470 475 480 Leu Asn Arg Leu Cys Ile Leu His Glu Lys Thr Pro Val Ser Glu Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu His Val Asp Glu Thr Tyr Val Pro Lys Pro Phe His Ala 515 520 525 Asp Ser Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Lys Glu 530 535 540 Lys Gln Val Lys Lys Gln Met Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Ser Glu Glu Gln Met Lys Thr Val Met Gly Asp Phe Ala 565 570 575 Ala Phe Leu Lys Lys Cys Cys Asp Ala Asp Asn Lys Glu Ala Cys Phe 580 585 590 Thr Glu Asp Gly Pro Lys Leu Val Ala Lys Cys Gln Ala Thr Leu Ala 595 600 605 101607PRTEquus caballus 101Met Lys Trp Val Thr Phe Val Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Leu Arg Arg Asp Thr His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Leu Gly Glu Lys His Phe Lys Gly Leu Val Leu 35 40 45 Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Lys Cys Ala Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr 115 120 125 His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135 140 Ala Gln Cys Ala Ala Phe Gln Glu Asp Pro Asp Lys Phe Leu Gly Lys 145 150 155 160 Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly Pro Glu 165 170 175 Leu Leu Phe His Ala Glu Glu Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185 190 Pro Ala Asp Asp Lys Leu Ala Cys Leu Ile Pro Lys Leu Asp Ala Leu 195 200 205 Lys Glu Arg Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215 220 Ser Phe Gln Asn Phe Gly Glu Arg Ala Val Lys Ala Trp Ser Val Ala 225 230 235 240 Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser Lys 245 250 255 Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285 Glu His Gln Asp Ser Ile Ser Gly Lys Leu Lys Ala Cys Cys Asp Lys 290 295 300 Pro Leu Leu Gln Lys Ser His Cys Ile Ala Glu Val Lys Glu Asp Asp 305 310 315 320 Leu Pro Ser Asp Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys 325 330 335 Glu Ile Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345 350 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360 365 Leu Leu Arg Ile Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys Cys Cys 370 375 380 Ala Glu Ala Asp Pro Pro Ala Cys Tyr Arg Thr Val Phe Asp Gln Phe 385 390 395 400 Thr Pro Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn Cys Asp 405 410 415 Leu Phe Glu Glu Val Gly Glu Tyr Asp Phe Gln Asn Ala Leu Ile Val 420 425 430 Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Ile Gly Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455 460 Glu Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Ile 485 490 495 Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Glu Leu Asp Glu Gly Tyr Val Pro Lys Glu Phe Lys Ala Glu 515 520 525 Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Asp Glu Lys 530 535 540 Gln Ile Lys Lys Gln Ser Ala Leu Ala Glu Leu Val Lys His Lys Pro 545 550 555 560 Lys Ala Thr Lys Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala 565 570 575 Phe Val Ala Lys Cys Cys Gly Arg Glu Asp Lys Glu Ala Cys Phe Ala 580 585 590 Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala 595 600 605 102609PRTHomo sapiens 102Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu 103600PRTMacca mulatta 103Leu Leu Phe Leu Phe Ser Ser Ala Tyr Ser Arg

Gly Val Phe Arg Arg 1 5 10 15 Asp Thr His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 20 25 30 Glu His Phe Lys Gly Leu Val Leu Val Ala Phe Ser Gln Tyr Leu Gln 35 40 45 Gln Cys Pro Phe Glu Glu His Val Lys Leu Val Asn Glu Val Thr Glu 50 55 60 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 65 70 75 80 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 85 90 95 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 100 105 110 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 115 120 125 Pro Pro Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 130 135 140 Asp Asn Glu Ala Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Val Ala Arg 145 150 155 160 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Ala Arg 165 170 175 Tyr Lys Ala Ala Phe Ala Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 180 185 190 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 195 200 205 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Asp 210 215 220 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Lys Phe Pro 225 230 235 240 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 245 250 255 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 260 265 270 Arg Ala Asp Leu Ala Lys Tyr Met Cys Glu Asn Gln Asp Ser Ile Ser 275 280 285 Ser Lys Leu Lys Glu Cys Cys Asp Lys Pro Leu Leu Glu Lys Ser His 290 295 300 Cys Leu Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 305 310 315 320 Leu Ala Ala Asp Tyr Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 325 330 335 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 340 345 350 Arg His Pro Asp Tyr Ser Val Met Leu Leu Leu Arg Leu Ala Lys Ala 355 360 365 Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 370 375 380 Cys Tyr Ala Lys Val Phe Asp Glu Phe Gln Pro Leu Val Glu Glu Pro 385 390 395 400 Gln Asn Leu Val Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 405 410 415 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 420 425 430 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 435 440 445 Val Gly Ala Lys Cys Cys Lys Leu Pro Glu Ala Lys Arg Met Pro Cys 450 455 460 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Arg Leu Cys Val Leu His 465 470 475 480 Glu Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu Ser 485 490 495 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Leu Asp Glu Ala 500 505 510 Tyr Val Pro Lys Ala Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 515 520 525 Met Cys Thr Leu Ser Glu Lys Glu Lys Gln Val Lys Lys Gln Thr Ala 530 535 540 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 545 550 555 560 Lys Gly Val Met Asp Asn Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 565 570 575 Ala Asp Asp Lys Glu Ala Cys Phe Ala Glu Glu Gly Pro Lys Phe Val 580 585 590 Ala Ala Ser Gln Ala Ala Leu Ala 595 600 104608PRTMus musculus 104Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala 1 5 10 15 Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala 50 55 60 Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala 130 135 140 Glu Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly 145 150 155 160 His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys 180 185 190 Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly 195 200 205 Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys 210 215 220 Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr 245 250 255 Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp 290 295 300 Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp 305 310 315 320 Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp 325 330 335 Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu 385 390 395 400 Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys 405 410 415 Asp Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460 Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile 465 470 475 480 Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His 485 490 495 Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525 Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu 530 535 540 Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala 565 570 575 Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe 580 585 590 Ser Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala 595 600 605 105607PRTSus scrofa 105Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr Tyr Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Gln Tyr Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln His Leu Gln Gln Cys Pro Tyr Glu Glu His Val 50 55 60 Lys Leu Val Arg Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Ser Leu Arg Glu His Tyr Gly Asp Leu Ala 100 105 110 Asp Cys Cys Glu Lys Glu Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asn Asp Asn Pro Asp Ile Pro Lys Leu Lys Pro Asp Pro Val 130 135 140 Ala Leu Cys Ala Asp Phe Gln Glu Asp Glu Gln Lys Phe Trp Gly Lys 145 150 155 160 Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170 175 Leu Leu Tyr Tyr Ala Ile Ile Tyr Lys Asp Val Phe Ser Glu Cys Cys 180 185 190 Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Ile Glu His Leu 195 200 205 Arg Glu Lys Val Leu Thr Ser Ala Ala Lys Gln Arg Leu Lys Cys Ala 210 215 220 Ser Ile Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Leu Ala 225 230 235 240 Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Thr Glu Ile Ser Lys 245 250 255 Ile Val Thr Asp Leu Ala Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285 Glu Asn Gln Asp Thr Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp Lys 290 295 300 Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Ala Lys Arg Asp Glu 305 310 315 320 Leu Pro Ala Asp Leu Asn Pro Leu Glu His Asp Phe Val Glu Asp Lys 325 330 335 Glu Val Cys Lys Asn Tyr Lys Glu Ala Lys His Val Phe Leu Gly Thr 340 345 350 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu 355 360 365 Leu Leu Arg Ile Ala Lys Ile Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375 380 Ala Lys Glu Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp Lys Phe 385 390 395 400 Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Ile Lys Gln Asn Cys Glu 405 410 415 Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val 420 425 430 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Val Ala Arg Lys Leu Gly Leu Val Gly Ser Arg Cys Cys Lys Arg Pro 450 455 460 Glu Glu Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Leu Val Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490 495 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu Gly 515 520 525 Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Asp Glu Lys 530 535 540 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro 545 550 555 560 His Ala Thr Glu Glu Gln Leu Arg Thr Val Leu Gly Asn Phe Ala Ala 565 570 575 Phe Val Gln Lys Cys Cys Ala Ala Pro Asp His Glu Ala Cys Phe Ala 580 585 590 Val Glu Gly Pro Lys Phe Val Ile Glu Ile Arg Gly Ile Leu Ala 595 600 605 106608PRTOryctolagus cuniculus 106Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Val Gly Glu Glu His Phe Ile Gly Leu Val Leu 35 40 45 Ile Thr Phe Ser Gln Tyr Leu Gln Lys Cys Pro Tyr Glu Glu His Ala 50 55 60 Lys Leu Val Lys Glu Val Thr Asp Leu Ala Lys Ala Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Asp Ile Phe Gly Asp 85 90 95 Lys Ile Cys Ala Leu Pro Ser Leu Arg Asp Thr Tyr Gly Asp Val Ala 100 105 110 Asp Cys Cys Glu Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu His 115 120 125 His Lys Asp Asp Lys Pro Asp Leu Pro Pro Phe Ala Arg Pro Glu Ala 130 135 140 Asp Val Leu Cys Lys Ala Phe His Asp Asp Glu Lys Ala Phe Phe Gly 145 150 155 160 His Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Gln Lys Tyr Lys Ala Ile Leu Thr Glu Cys 180 185 190 Cys Glu Ala Ala Asp Lys Gly Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200 205 Leu Glu Gly Lys Ser Leu Ile Ser Ala Ala Gln Glu Arg Leu Arg Cys 210 215 220 Ala Ser Ile Gln Lys Phe Gly Asp Arg Ala Tyr Lys Ala Trp Ala Leu 225 230 235 240 Val Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Thr Asp Ile Ser 245 250 255 Lys Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met 275 280 285 Cys Glu His Gln Glu Thr Ile Ser Ser His Leu Lys Glu Cys Cys Asp 290 295 300 Lys Pro Ile Leu Glu Lys Ala His Cys Ile Tyr Gly Leu His Asn Asp 305 310 315 320 Glu Thr Pro Ala Gly Leu Pro Ala Val Ala Glu Glu Phe Val Glu Asp 325 330 335 Lys Asp Val Cys Lys Asn Tyr Glu Glu Ala Lys Asp Leu Phe Leu Gly 340 345 350 Lys Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Gly Lys Ala Tyr Glu Ala Thr Leu Lys Lys Cys 370 375 380 Cys Ala Thr Asp Asp Pro His Ala Cys Tyr Ala Lys Val Leu Asp Glu 385 390 395 400 Phe Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Val Lys Gln Asn Cys 405 410 415 Glu Leu Tyr Glu Gln Leu Gly Asp Tyr Asn Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ile Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Glu Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys 485

490 495 Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asp Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Gly Pro Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Thr Glu 530 535 540 Arg Lys Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro His Ala Thr Asn Asp Gln Leu Lys Thr Val Val Gly Glu Phe Thr 565 570 575 Ala Leu Leu Asp Lys Cys Cys Ser Ala Glu Asp Lys Glu Ala Cys Phe 580 585 590 Ala Val Glu Gly Pro Lys Leu Val Glu Ser Ser Lys Ala Thr Leu Gly 595 600 605 107608PRTRattus norvegicus 107Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Ile Ser Gly Ser Ala 1 5 10 15 Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Pro Tyr Glu Glu His Ile 50 55 60 Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Asn Ala Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Lys Leu Arg Asp Asn Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Gln Arg Pro Glu Ala 130 135 140 Glu Ala Met Cys Thr Ser Phe Gln Glu Asn Pro Thr Ser Phe Leu Gly 145 150 155 160 His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Asn Glu Val Leu Thr Gln Cys 180 185 190 Cys Thr Glu Ser Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200 205 Val Lys Glu Lys Ala Leu Val Ala Ala Val Arg Gln Arg Met Lys Cys 210 215 220 Ser Ser Met Gln Arg Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Met Ser Gln Arg Phe Pro Asn Ala Glu Phe Ala Glu Ile Thr 245 250 255 Lys Leu Ala Thr Asp Val Thr Lys Ile Asn Lys Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Ala Cys Cys Asp 290 295 300 Lys Pro Val Leu Gln Lys Ser Gln Cys Leu Ala Glu Ile Glu His Asp 305 310 315 320 Asn Ile Pro Ala Asp Leu Pro Ser Ile Ala Ala Asp Phe Val Glu Asp 325 330 335 Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser 355 360 365 Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Glu Gly Asp Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu 385 390 395 400 Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys 405 410 415 Glu Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Val Leu 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460 Pro Glu Ala Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile 465 470 475 480 Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys 485 490 495 Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525 Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Asp Lys Glu 530 535 540 Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Glu Asp Gln Leu Lys Thr Val Met Gly Asp Phe Ala 565 570 575 Gln Phe Val Asp Lys Cys Cys Lys Ala Ala Asp Lys Asp Asn Cys Phe 580 585 590 Ala Thr Glu Gly Pro Asn Leu Val Ala Arg Ser Lys Glu Ala Leu Ala 595 600 605 108607PRTOvis aries 108Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gln Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val 50 55 60 Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser His Ala Gly Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Glu Leu Cys Lys Val Ala Thr Leu Arg Glu Thr Tyr Gly Asp Met Ala 100 105 110 Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Asn 115 120 125 His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135 140 Thr Leu Cys Ala Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys 145 150 155 160 Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170 175 Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys 180 185 190 Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp Ala Met 195 200 205 Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala 210 215 220 Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala 225 230 235 240 Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Thr Asp Val Thr Lys 245 250 255 Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285 Asp His Gln Asp Ala Leu Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys 290 295 300 Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp Ala 305 310 315 320 Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys 325 330 335 Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly Ser 340 345 350 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val 355 360 365 Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375 380 Ala Lys Glu Asp Pro His Ala Cys Tyr Ala Thr Val Phe Asp Lys Leu 385 390 395 400 Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu 405 410 415 Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val 420 425 430 Arg Tyr Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Cys Ala Lys Pro 450 455 460 Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490 495 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510 Asp Leu Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys 515 520 525 Phe Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys 530 535 540 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro 545 550 555 560 Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala 565 570 575 Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Gly Cys Phe Val 580 585 590 Leu Glu Gly Pro Lys Leu Val Ala Ser Thr Gln Ala Ala Leu Ala 595 600 605 109609PRTHomo sapiens 109Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu 110609PRTPongo abelii 110Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Lys Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Val Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu Lys Ser His Cys Leu Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu

Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Glu Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Pro Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Asp Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Thr Val Met Glu Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600 605 Leu 111615PRTGallus gallus 111Met Lys Trp Val Thr Leu Ile Ser Phe Ile Phe Leu Phe Ser Ser Ala 1 5 10 15 Thr Ser Arg Asn Leu Gln Arg Phe Ala Arg Asp Ala Glu His Lys Ser 20 25 30 Glu Ile Ala His Arg Tyr Asn Asp Leu Lys Glu Glu Thr Phe Lys Ala 35 40 45 Val Ala Met Ile Thr Phe Ala Gln Tyr Leu Gln Arg Cys Ser Tyr Glu 50 55 60 Gly Leu Ser Lys Leu Val Lys Asp Val Val Asp Leu Ala Gln Lys Cys 65 70 75 80 Val Ala Asn Glu Asp Ala Pro Glu Cys Ser Lys Pro Leu Pro Ser Ile 85 90 95 Ile Leu Asp Glu Ile Cys Gln Val Glu Lys Leu Arg Asp Ser Tyr Gly 100 105 110 Ala Met Ala Asp Cys Cys Ser Lys Ala Asp Pro Glu Arg Asn Glu Cys 115 120 125 Phe Leu Ser Phe Lys Val Ser Gln Pro Asp Phe Val Gln Pro Tyr Gln 130 135 140 Arg Pro Ala Ser Asp Val Ile Cys Gln Glu Tyr Gln Asp Asn Arg Val 145 150 155 160 Ser Phe Leu Gly His Phe Ile Tyr Ser Val Ala Arg Arg His Pro Phe 165 170 175 Leu Tyr Ala Pro Ala Ile Leu Ser Phe Ala Val Asp Phe Glu His Ala 180 185 190 Leu Gln Ser Cys Cys Lys Glu Ser Asp Val Gly Ala Cys Leu Asp Thr 195 200 205 Lys Glu Ile Val Met Arg Glu Lys Ala Lys Gly Val Ser Val Lys Gln 210 215 220 Gln Tyr Phe Cys Gly Ile Leu Lys Gln Phe Gly Asp Arg Val Phe Gln 225 230 235 240 Ala Arg Gln Leu Ile Tyr Leu Ser Gln Lys Tyr Pro Lys Ala Pro Phe 245 250 255 Ser Glu Val Ser Lys Phe Val His Asp Ser Ile Gly Val His Lys Glu 260 265 270 Cys Cys Glu Gly Asp Met Val Glu Cys Met Asp Asp Met Ala Arg Met 275 280 285 Met Ser Asn Leu Cys Ser Gln Gln Asp Val Phe Ser Gly Lys Ile Lys 290 295 300 Asp Cys Cys Glu Lys Pro Ile Val Glu Arg Ser Gln Cys Ile Met Glu 305 310 315 320 Ala Glu Phe Asp Glu Lys Pro Ala Asp Leu Pro Ser Leu Val Glu Lys 325 330 335 Tyr Ile Glu Asp Lys Glu Val Cys Lys Ser Phe Glu Ala Gly His Asp 340 345 350 Ala Phe Met Ala Glu Phe Val Tyr Glu Tyr Ser Arg Arg His Pro Glu 355 360 365 Phe Ser Ile Gln Leu Ile Met Arg Ile Ala Lys Gly Tyr Glu Ser Leu 370 375 380 Leu Glu Lys Cys Cys Lys Thr Asp Asn Pro Ala Glu Cys Tyr Ala Asn 385 390 395 400 Ala Gln Glu Gln Leu Asn Gln His Ile Lys Glu Thr Gln Asp Val Val 405 410 415 Lys Thr Asn Cys Asp Leu Leu His Asp His Gly Glu Ala Asp Phe Leu 420 425 430 Lys Ser Ile Leu Ile Arg Tyr Thr Lys Lys Met Pro Gln Val Pro Thr 435 440 445 Asp Leu Leu Leu Glu Thr Gly Lys Lys Met Thr Thr Ile Gly Thr Lys 450 455 460 Cys Cys Gln Leu Gly Glu Asp Arg Arg Met Ala Cys Ser Glu Gly Tyr 465 470 475 480 Leu Ser Ile Val Ile His Asp Thr Cys Arg Lys Gln Glu Thr Thr Pro 485 490 495 Ile Asn Asp Asn Val Ser Gln Cys Cys Ser Gln Leu Tyr Ala Asn Arg 500 505 510 Arg Pro Cys Phe Thr Ala Met Gly Val Asp Thr Lys Tyr Val Pro Pro 515 520 525 Pro Phe Asn Pro Asp Met Phe Ser Phe Asp Glu Lys Leu Cys Ser Ala 530 535 540 Pro Ala Glu Glu Arg Glu Val Gly Gln Met Lys Leu Leu Ile Asn Leu 545 550 555 560 Ile Lys Arg Lys Pro Gln Met Thr Glu Glu Gln Ile Lys Thr Ile Ala 565 570 575 Asp Gly Phe Thr Ala Met Val Asp Lys Cys Cys Lys Gln Ser Asp Ile 580 585 590 Asn Thr Cys Phe Gly Glu Glu Gly Ala Asn Leu Ile Val Gln Ser Arg 595 600 605 Ala Thr Leu Gly Ile Gly Ala 610 615 112386PRTGallus gallus 112Met Gly Ser Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe 1 5 10 15 Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro 20 25 30 Ile Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 35 40 45 Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro 50 55 60 Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn Val 65 70 75 80 His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp 85 90 95 Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Arg Tyr 100 105 110 Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly 115 120 125 Gly Leu Glu Pro Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Glu 130 135 140 Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn 145 150 155 160 Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val 165 170 175 Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu 180 185 190 Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro 195 200 205 Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser Met Ala 210 215 220 Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met 225 230 235 240 Ser Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 245 250 255 Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 260 265 270 Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 275 280 285 Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 290 295 300 Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu 305 310 315 320 Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 325 330 335 Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala 340 345 350 Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His Pro Phe Leu Phe Cys 355 360 365 Ile Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val 370 375 380 Ser Pro 385 113386PRTMaleagris gallopavo 113Met Gly Ser Ile Gly Ala Val Ser Met Glu Phe Cys Phe Asp Val Phe 1 5 10 15 Lys Glu Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Ser Pro 20 25 30 Phe Thr Ile Ile Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 35 40 45 Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro 50 55 60 Gly Phe Gly Asp Ser Val Glu Ala Gln Cys Gly Thr Ser Val Asn Val 65 70 75 80 His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys Pro Asn Asp 85 90 95 Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala Glu Glu Thr Tyr 100 105 110 Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val Lys Glu Leu Tyr Arg Gly 115 120 125 Gly Leu Glu Ser Ile Asn Phe Gln Thr Ala Ala Asp Gln Ala Arg Gly 130 135 140 Leu Ile Asn Ser Trp Val Glu Ser Gln Thr Asn Gly Met Ile Lys Asn 145 150 155 160 Val Leu Gln Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val 165 170 175 Asn Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Ala Phe Lys Asp Glu 180 185 190 Asp Thr Gln Ala Ile Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro 195 200 205 Val Gln Met Met Tyr Gln Ile Gly Leu Phe Lys Val Ala Ser Met Ala 210 215 220 Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala Ser Gly Thr Met 225 230 235 240 Ser Met Trp Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 245 250 255 Glu Thr Thr Ile Ser Phe Glu Lys Met Thr Glu Trp Ile Ser Ser Asn 260 265 270 Ile Met Glu Glu Arg Arg Ile Lys Val Tyr Leu Pro Arg Met Lys Met 275 280 285 Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 290 295 300 Asp Leu Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Gly 305 310 315 320 Ser Leu Lys Ile Ser Gln Ala Ala His Ala Ala Tyr Ala Glu Ile Tyr 325 330 335 Glu Ala Gly Arg Glu Val Ile Gly Ser Ala Glu Ala Gly Ala Asp Ala 340 345 350 Thr Ser Val Ser Glu Glu Phe Arg Val Asp His Pro Phe Leu Tyr Cys 355 360 365 Ile Lys His Asn Leu Thr Asn Ser Ile Leu Phe Phe Gly Arg Cys Ile 370 375 380 Ser Pro 385 114551PRTPetromyzon marinus 114Thr Met Gly Asp Cys Cys Gly Lys Glu Asn Ala Ala Gly Cys Leu Leu 1 5 10 15 His His Arg Tyr Leu Phe Gln Asp Glu Leu Cys Glu Gly Val Ser Ser 20 25 30 Ile Pro Ser Ala Ala Ser Cys Cys Ser Leu Ala Asn Glu Glu Asp Arg 35 40 45 Ala Asp Cys Leu Val Ser Leu Arg Gly Asn Leu Ser Ile His Ser Val 50 55 60 Pro Leu Ala Pro Ala Ser Gln Leu Cys His Asp Arg Arg Trp Lys Ser 65 70 75 80 His Glu Ser Phe Ala Ser Leu Leu Trp Glu Phe Gly Arg Arg His Pro 85 90 95 Arg Ala Ala Asp Ser Gln Val Glu Glu Leu Ala Glu Arg Phe Ser Lys 100 105 110 Ile Gly Asp Ala Cys Cys Asp Leu Ala Asp Glu Lys Glu Cys Ile Thr 115 120 125 Arg Gly Arg Glu Ala Ile His Gln Glu Val Ser Ala Ala Tyr Ala Asp 130 135 140 Ala Ala Gln Leu Cys Ser Ser Leu Gln Ala Leu Gly Ala Gln Lys Phe 145 150 155 160 Leu Gly Arg Met Val Leu Val Phe Ser Gln Arg Ala Pro Asn Ala Thr 165 170 175 Phe Asp Gln Ile Ser Lys Leu Ser His Arg Phe His Ser Tyr Ala Gln 180 185 190 Thr Cys Cys Gly Glu Gly Trp Ser Pro Gly Cys Phe Ala Glu Gln Arg 195 200 205 His Leu Ile His Asp Glu Met Cys His Asp Met Glu Ala Leu Ser Arg 210 215 220 Val Pro Ala Met Ala Lys Cys Cys Gln Ile Ser Gly Ser Ala Arg Ala 225 230 235 240 Lys Cys Met Glu Thr Ile Pro Arg Gly Lys Pro Val Leu Asp Val Ala 245 250 255 Leu Ala Arg Phe Asp Gly His Lys Val Cys Gln Met Asn Ala Glu Ala 260 265 270 Pro Gln Glu Leu Leu Gly Arg Met Leu Tyr Glu Phe Gly Arg Arg His 275 280 285 Thr Asp Ala Ser Val Gly Glu Ala Lys Lys Ile Ile Thr Glu Trp Met 290 295 300 Asp Gly Val Lys Asp Cys Cys Ala Gly Asn His Ser Glu Glu Gln Ala 305 310 315 320 Cys Leu Val Ser Lys Lys Ala Ala Ile Ser Val Lys Ile Gly Glu Glu 325 330 335 Gln Ala Lys Ser His Lys Ile Cys Glu Gln Leu Gln Lys Asp Gly His 340 345 350 Glu Val Phe Glu Glu Met Val Leu Ile Asp Phe Ala Ile Glu Ala Arg 355 360 365 Thr Leu Ser Leu Asp Lys Val Val Glu Phe Ala His Arg Tyr Thr His 370 375 380 His Ala Ile Arg Cys Cys Ala His Gln Ala His Cys Leu Leu Asp Glu 385 390 395 400 Asn Leu His Leu Phe Ser Ser Leu Cys Ser Asp Leu Ser Tyr Leu Ala 405 410 415 Ala His Asp Gly Tyr Arg Lys Cys Cys Arg Leu Ala Pro Ser Glu Ala 420 425 430 Val Ser Cys His Val Glu His Glu Arg Ala His Glu Ala Glu Arg Ala 435 440 445 Thr Glu Glu Val Glu Asn His Gly Lys Glu Arg Val Glu His Gln Ala 450 455 460 Lys Val Glu Ala Val Glu Ala Val Glu Ala Pro Phe Ala Glu Glu Gly 465 470 475 480 Ala Ala Arg Ser Cys Leu Arg Phe Arg Gln Leu Pro Gly Lys Tyr Leu 485 490 495 Gln Arg Leu Leu Tyr Lys Ala Ala His Gln Ala Pro Ala Gly Val Asp 500 505 510 His Ser Arg Ile Arg Leu Gln Val His His Phe Val Glu Val Thr Ala 515 520 525 Lys Cys Cys Arg Ala Tyr Asp Lys Ser Glu Cys Phe Ser His Glu Ile 530 535 540 Lys Glu Met Lys Asn Ser Pro 545 550 1151423PRTPetromyzon marinus 115Met Gly Lys Ala Met Leu Lys Leu Cys Ile Thr Leu Met Val Leu Val 1 5 10 15 Phe Ser Gly Thr Ala Glu Ser Lys Gly Val Met Arg Arg Glu Asp Glu 20 25 30 Ser Phe Pro His Leu Lys Ser Arg Leu Cys Gly Gly Leu Asn Gly Leu 35 40 45 Gly Glu Asp Ala Tyr Arg Ser His Cys Val Val Tyr Tyr Thr Lys Arg 50 55 60 Met Gly Val Val Ser Leu Asp His Val Glu Glu Leu Ala Asn His Cys 65 70 75 80 Leu Arg Ile Val Lys Gln Cys Cys Ala Glu Gly Ala Ala Asp Asp Cys 85 90 95 Leu Gln

Thr Glu Leu Ala Ala Val Gln Glu Gln Val Cys Thr Arg Met 100 105 110 Ser Glu Ala Lys Asp Val Pro Leu Val Gly Arg Cys Cys Ala Leu Ala 115 120 125 Gly Ser Glu Arg His Asp Cys Phe His His Ala Gly Gly Val Ala Glu 130 135 140 Gly Glu Gly Ala Trp Pro His Ala Leu Pro Val Thr Ser Pro Pro Glu 145 150 155 160 Tyr Asp Ser Val Thr Val Cys Ala Leu His Ala Thr Ala Asn Ala Arg 165 170 175 Leu Tyr Asp Thr Leu Leu Trp Glu Phe Ser Arg Arg Tyr Pro Ser Ala 180 185 190 Ser Asp Ser His Leu Ile Ala Leu Ala Asn Glu Phe Ile Thr Gly Leu 195 200 205 Thr Thr Cys Cys Leu Val Glu Glu Glu His Gly Ala Cys Leu Ala Thr 210 215 220 Leu Arg Glu Asp Phe Lys His Lys Leu Thr Glu Ala Ser His Lys Ser 225 230 235 240 Gln Asn Leu Cys Lys Ala Leu Lys Ser Leu Gly Lys Glu Lys Phe Glu 245 250 255 Asp Arg Ile Ile Val Arg Phe Thr Gln Arg Ala Pro Gln Ala Pro Phe 260 265 270 Glu Leu Ile Gln Lys Leu Ala His Arg Phe Glu Val Leu Ala Glu Lys 275 280 285 Cys Cys Glu Leu Gly His Ser Asp Arg Cys Leu Val Glu Glu Arg Tyr 290 295 300 Thr Val Asp Asp Glu Leu Cys Leu Glu Gln Ser Phe Val Ala Thr Cys 305 310 315 320 Pro Arg Leu Ser Ser Cys Cys Ser Leu Ser Gly Ser Ser Arg Ala Gln 325 330 335 Cys Leu Glu Thr Val Pro Val Leu Glu Thr Ser Asp Lys Ala Ser Pro 340 345 350 Ala Thr Pro Thr Leu Pro Ile Ser Glu Gln Cys Thr Leu Trp Ala Gly 355 360 365 Lys Pro Val Glu Phe His Lys Arg Val Val Trp Gln Ile Ser His Arg 370 375 380 Tyr Pro Thr Thr Gly Val Ala Gln Val Glu Ala Leu Ala His His Tyr 385 390 395 400 Leu Glu His Leu Thr Ile Cys Cys Ala Ser Glu Asp Lys Asp Thr Cys 405 410 415 Ile Ala Thr Glu Val Ala Glu Phe Lys Ser Glu Val Glu Lys Val His 420 425 430 Thr Lys Ser Asp Trp Trp Cys Arg Met Ser Asp Leu Leu Gly Thr Asp 435 440 445 Arg Phe Asn Leu Leu Leu Ile Val Thr Tyr Ser Gln Arg Val Pro Gln 450 455 460 Ala Thr Phe Glu Gln Val Glu Glu Ile Ser His His Phe Ala Leu Ile 465 470 475 480 Thr Arg Lys Cys Cys Ser His Arg Lys Asn Gly Ser Cys Phe Leu Glu 485 490 495 Glu Arg Tyr Ala Leu His Asp Ala Ile Cys Arg Asp Glu Ala Trp Leu 500 505 510 Ser Gly Leu Ala Glu Val Ser Arg Cys Cys Ala Met Asp Gly Arg Ala 515 520 525 Arg Ile Leu Cys Phe Asp Glu Leu Ser Ser His Leu Asn Ala Ser Val 530 535 540 Glu Glu Arg Pro Glu Leu Cys Ser Thr Ser Leu Cys Ser Lys Tyr His 545 550 555 560 Asp Leu Gly Phe Glu Phe Lys Gln Arg Val Ala Tyr Gly Phe Gly Gln 565 570 575 Arg Phe Pro Lys Ala Ala Met Gly Gln Met Arg Asp Leu Ile Ser Lys 580 585 590 Tyr Leu Ala Met Val Gln Arg Cys Cys Asp Ala Met Ser Asp Phe Lys 595 600 605 Met Asp Val Glu Glu Val Glu Leu Arg Ala His Arg Leu Cys Leu Asp 610 615 620 Ala His Gln Leu Gly Glu Glu Lys Leu Ala Asp Arg Ile Met Ile Gly 625 630 635 640 Leu Ala Gln Arg Ile Ser Val Ala Ser Phe Val Asn Ile Ser Ser Val 645 650 655 Ala Leu His Phe Ala Gln Ser Val Ile Lys Cys Cys Asp Ala Asp His 660 665 670 Glu Lys Thr Cys Phe Met Glu Gln Glu Phe Ala Leu Glu Asp Gln Val 675 680 685 Cys Ser Asp Ser Glu Ala Leu Ser His Ile Pro Ser Val Ser Arg Cys 690 695 700 Cys Glu Leu His Pro Phe Asp Arg Ser Val Cys Phe His Ser Leu Arg 705 710 715 720 Ser Thr Gln Ala Ser Thr Leu Ala Ser Thr His Val Ala Val Gly Lys 725 730 735 Asp Asp Ser Leu Pro Gly His Val Glu Glu Cys Gln Ala Phe Ala Ser 740 745 750 Gly Asn His Ser Leu Thr Asp Gln Val Met Phe Glu Phe Ala Arg Arg 755 760 765 His Pro Arg Ala Ser Val Ser Gln Val Glu Ser Leu Ala Arg Leu Tyr 770 775 780 Ser Glu Leu Ala Arg Ala Cys Cys Ala Leu Thr Asp Ala Asp Gln Glu 785 790 795 800 Ser Cys Leu His Thr Ala Arg Ser Gln Ala Arg Gln Glu Ala Leu Lys 805 810 815 Ser Leu Gln Arg Ser Glu Arg Ile Cys Asn Thr Leu Ser Ala Ile Gly 820 825 830 Lys Glu Lys Phe Glu Asp Arg Ile Val Ile Ala Leu Ser Gln Lys Ala 835 840 845 Thr Asp Ala Ser Phe Glu Gln Ile Leu Glu Ile Ala Asn Arg Met Ser 850 855 860 Arg Gly Leu Ala Arg Cys Cys Glu Gln Gly Asn Asn Val Gly Cys Leu 865 870 875 880 Met Asp His Arg His Ala Leu His Glu Ala Ile Cys Ser Thr Pro Asp 885 890 895 Gly Ser Leu Pro Gln Ser Val Ala Ala Cys Cys Asn Thr Ser Asn Thr 900 905 910 Ser Thr Thr Thr Ser Thr Thr Thr Ser Thr Thr Thr Ser Thr Thr Thr 915 920 925 Ser Thr Thr Thr Ser Thr Thr Ser Thr Thr Thr Ala Ala Glu Ile Arg 930 935 940 Asp Ser Cys Phe Asp Asn Leu Gln Ala Asn Val Ser Arg Ala His Ala 945 950 955 960 Pro Phe Tyr Ser Asn Ser Gln Leu Cys Leu Met Lys Leu Arg Thr Pro 965 970 975 His Arg Phe Leu Glu Arg Phe Leu Trp Glu Phe Gly Arg Arg His Pro 980 985 990 Gln Ala Ala Leu Ser Gln Val Glu Glu Leu Ala Glu Met Tyr Val Lys 995 1000 1005 Met Thr Asp Ser Cys Cys Gly Lys Leu His Ser Lys Ser Cys Phe 1010 1015 1020 Thr Glu Gln Arg His Thr Ile His Met Glu Ile Arg His Ala Tyr 1025 1030 1035 Ala Glu Val Gln His Ile Cys Gly Ser Leu His Ser Arg Gly Glu 1040 1045 1050 Glu Thr Phe Ile Gln Arg Glu Val Thr Leu Leu Ser Gln Lys Ala 1055 1060 1065 Pro Asn Ala Ser Phe Glu Lys Val Ser Gln Leu Ala Arg His Phe 1070 1075 1080 Leu Ser Leu Ala Lys Lys Cys Cys Ala Pro Asp His Ala Ala Gly 1085 1090 1095 Cys Phe Leu Glu Glu Pro Tyr Ala Ile His Asp Glu Val Cys Arg 1100 1105 1110 Asp Asp Glu Val Val Asp Gln Val Gly Gly Leu Ala Thr Cys Cys 1115 1120 1125 Arg Met Ser Gly Thr Ser Arg Ala Lys Cys Leu Ala Gln Leu Pro 1130 1135 1140 Arg Asp Leu Gly Arg His Gly Asn Arg Glu Thr Pro Glu Phe Asp 1145 1150 1155 Glu Leu Lys Ile Cys Glu Leu Arg Arg Asp Asn Pro Ala Val Leu 1160 1165 1170 Met Glu Lys Ile Leu Tyr Glu Phe Gly Arg Arg His Ser Asp Ser 1175 1180 1185 Ala Val Ser Glu Val Lys Asn Phe Ala Gln Lys Phe Ser His Ser 1190 1195 1200 Val Thr Glu Cys Cys Thr Ser Glu Lys Thr His Glu Cys Phe Val 1205 1210 1215 Glu Lys Arg Ala Ala Ile Glu Lys Val Ile Lys Asp Glu Glu Ala 1220 1225 1230 Lys Gly Asn Leu Thr Cys Gln Arg Leu Lys Ala Gln Gly Val Glu 1235 1240 1245 His Phe Glu Gln Leu Val Ile Leu Asn Phe Ala Arg Ala Ala Lys 1250 1255 1260 Ser Leu Pro Met Glu Lys Val Val Glu Phe Ala His Arg Phe Thr 1265 1270 1275 Arg Ile Ala Gly Gln Cys Cys Glu His Asp Thr His Cys Leu Ile 1280 1285 1290 Asp Glu Ser Phe His Leu His Ala Glu Met Cys Gly Asp His Gly 1295 1300 1305 Tyr Ile Met Ala His Pro Gly Val Ala Asn Cys Cys Lys Ser Asp 1310 1315 1320 Val Ser Glu Gln Gly Thr Cys Phe Lys Ile His Glu Asp Val His 1325 1330 1335 His Ala Glu Glu Ile Leu Ser Lys Asp Val Ser Pro Ala His Pro 1340 1345 1350 Thr Ala Glu Arg Val Cys Leu Arg Tyr Arg Gln Phe Pro Glu Lys 1355 1360 1365 Phe Ile Asn Leu Ala Leu Phe Glu Leu Val His Arg Leu Pro Leu 1370 1375 1380 Leu Glu Ser Ser Val Leu Arg Arg Lys Ala Leu Ala Tyr Thr Gly 1385 1390 1395 Phe Thr Asp Asp Cys Cys Arg Ala Val Asp Lys Thr Ala Cys Phe 1400 1405 1410 Thr Glu Lys Leu Glu Ala Ile Lys Ser Ser 1415 1420 116382PRTRana catesbeiana 116Lys Cys Arg Ile Ile Arg Glu Phe Pro Asp Ile Val Phe Lys Gly Leu 1 5 10 15 Thr Leu Val Gln Val Ser Gln Lys Phe Gly Lys Ala Gly Phe Glu Asp 20 25 30 Val Lys Lys Val Thr Glu Glu Ile Val His Leu Asn Glu Asp Cys Cys 35 40 45 Lys Gly Asp Ala Val Glu Cys Met Met Glu Arg Met Glu Ala Thr Asp 50 55 60 His Ile Cys Glu Ala Lys Asp Lys Leu Ser Ser Lys Leu Ala Asp Cys 65 70 75 80 Cys Ala Lys Ser Ile Leu Glu Arg Thr Pro Cys Leu Leu Ala Leu Pro 85 90 95 Asn Asp Glu Ser Asp Leu Ser Lys Glu Leu Lys Asn Tyr Tyr Glu Asp 100 105 110 Glu Arg Val Cys Glu Asn Tyr Lys Lys Asp Lys Leu Leu Phe Leu Ala 115 120 125 His Phe Thr His Asp Tyr Ala Arg Ser His Gln Glu Ser Ser Pro Gln 130 135 140 Ser Cys Leu Arg Val Ser Lys Gly Phe Glu Gly Leu Leu Glu Lys Cys 145 150 155 160 Cys Ala Ser Glu Asn His Ala Glu Cys Leu Lys Gln Ala Pro Ile Leu 165 170 175 Leu Glu Ala Ala Leu Lys Glu Ile Glu Glu Leu Arg Lys Gln Asn Cys 180 185 190 Gly Ala Leu Gln Leu Leu Gly Phe Arg Asp Tyr Asn Ile Gln Leu Leu 195 200 205 Phe Arg Tyr Phe Phe Lys Met Pro Gln Val Thr Ala Pro Thr Leu Val 210 215 220 Glu Leu Ala Gly Arg Met Thr Lys Val Ala Val Tyr Cys Cys Gly Leu 225 230 235 240 Ala Glu Asn Lys Gln Gln Thr Cys Ala Glu Glu Lys Leu Asp Ile Leu 245 250 255 Leu Gly Glu Met Cys Glu Lys Glu Lys His Thr Phe Val Asn Asp Asn 260 265 270 Val Arg His Cys Cys Val Asp Ser Tyr Ala Asn Arg Arg Lys Cys Phe 275 280 285 Thr Asp Leu Gln Arg Tyr Pro Asn Tyr Val Ala Pro Lys Trp Asp Glu 290 295 300 Ser Lys Leu His Phe Asn Glu Asp Leu Cys Lys Gly Ser Glu Asp Asp 305 310 315 320 Gln Ile Lys Lys Lys Leu Glu Val Leu Val Glu Tyr Met Lys Met Lys 325 330 335 Pro Asp Cys Gly Pro Glu Lys Leu Lys Glu Val Val Glu Ala Phe Arg 340 345 350 Lys Ile Asp Ile Lys Cys Cys Ala Ala Glu Asp His Gln Lys Cys Phe 355 360 365 Asp Asp Glu Lys Ala Gly Leu Leu Gln Ile Ile Glu Ala His 370 375 380 117603PRTRana shqiperica 117Lys Trp Ala Thr Leu Ile Cys Leu Phe Ile Leu Ser Ile Thr Thr Glu 1 5 10 15 Ser Arg His Leu Gln Lys Arg His His Glu Glu His Pro Arg Ile Ile 20 25 30 Asn Asp Ile Val Lys Ala Val Gly Lys Pro Ala Val Glu Lys Leu Val 35 40 45 Leu Val Met Val Ala Gln Asp Phe Glu Lys Cys Ser Leu Asp Glu His 50 55 60 Leu Lys Val Gln Ala Lys Ile Ile Glu Ala Val Asp Asn Cys Glu Lys 65 70 75 80 His Pro Glu Glu Ala Glu Cys Lys Lys Pro Ala Ile Glu Leu Tyr His 85 90 95 Asp Ile Val Cys Lys Glu Glu Asp Ile Asp Gln Leu Tyr Pro Trp Thr 100 105 110 Thr Glu Cys Cys Gly Lys Ala Glu Ala Glu Arg Thr Lys Cys Phe Tyr 115 120 125 Glu His Arg Glu Val Arg Val Glu Glu Tyr Lys Ile Pro Asn Ile Glu 130 135 140 Glu Ser Cys Lys Glu His Lys Glu His Pro Gln Arg Ala Phe Ser Tyr 145 150 155 160 Tyr Leu Ser Asn Ile Ala Lys Arg His Ser Lys Leu Tyr Pro Pro Ala 165 170 175 Val Leu Gly Phe Ala Ile Gln Tyr Asn Glu Ile Thr Thr Glu Cys Cys 180 185 190 Ala Ala Glu Asp Lys Ala Lys Cys Phe Gly Glu Arg Met Pro Gln Val 195 200 205 Lys Lys Leu Thr Asn Tyr Leu Glu Asp Lys His Lys Gln Lys Cys Arg 210 215 220 Val Leu Lys Glu Phe Pro Glu Arg Val Ser Gln Ala Leu Thr Leu Val 225 230 235 240 Gln Val Ser Gln Arg Phe Gly Asn Ala Lys Tyr Asp Asp Val Glu Lys 245 250 255 Val Thr Ile Glu Ile Ala His Leu Asn Glu Asp Cys Cys Lys Gly Asp 260 265 270 Ala Val Glu Cys Met Ile Glu Arg Met Glu Ala Thr Glu His Ile Cys 275 280 285 Leu Ala Lys Glu Lys Leu Ser Ser Lys Leu Ser Asp Cys Cys Ala Lys 290 295 300 Gly Val Leu Glu Arg Thr Pro Cys Ile Leu Ala Leu Pro Asn Glu Glu 305 310 315 320 Pro Asp Leu Pro Ile Glu Leu Lys Glu Tyr Tyr Glu Asp Glu His Val 325 330 335 Cys Glu Asn Tyr Gln Lys Asp Lys Arg Lys Tyr Leu Ala His Phe Thr 340 345 350 His Asp Tyr Ser Arg Ser His Gln Glu Ser Ser Pro Gln Ser Cys Leu 355 360 365 Arg Val Ser Arg Gly Phe Glu Met Leu Leu Glu Lys Cys Cys Ala Ser 370 375 380 Ala Asn Ser Ala Glu Cys Leu Lys Asp Ala Pro Lys Leu Leu Glu Ala 385 390 395 400 Ala Leu Lys Glu Asn Glu Glu Ile Ser Lys Gln Asn Cys Gly Ala Leu 405 410 415 Glu Lys Leu Gly Phe Asn Asp Phe Tyr Ile Gln Leu Leu Val Arg Tyr 420 425 430 Phe Gly Lys Met Pro Gln Val Thr Ala Gln Thr Leu Val Glu Leu Thr 435 440 445 Gly Arg Met Ala Lys Ile Gly Val Tyr Cys Cys Gly Leu Pro Asp Asn 450 455 460 Lys Lys Gln Pro Cys Ala Glu Glu Lys Leu Asp Ile Leu Leu Gly Glu 465 470 475 480 Met Cys Glu Arg Glu Lys Lys Thr Phe Ile Asn Asp Asn Val His His 485 490 495 Cys Cys Val Asp Ser Tyr Ala Asn Arg Arg Pro Cys Phe Thr Lys Leu 500 505 510 Gly Pro Tyr Ala Asn Tyr Glu Ala Pro Val Trp Asp Glu Ser Lys Leu 515 520 525 His Phe Thr Ala Asp Met Cys Lys Gly Ser Ala Asp Asp Gln Leu Lys 530 535 540 Thr Lys Leu Val Leu Leu Val Glu Phe Leu Lys Met Lys Pro Thr Cys 545 550 555 560 Gly Lys Glu Lys Leu Thr Glu Val Ile Glu Ser Phe Arg Lys Thr Val 565

570 575 Val Glu Cys Cys Ala Ala Glu Asn Gln Gln Ala Cys Phe Asp Glu Lys 580 585 590 Lys Gly Gly Leu His Glu Ile Ile Lys Asp His 595 600 118608PRTXenopus laevis 118Met Lys Trp Ile Thr Leu Ile Cys Leu Leu Ile Ser Ser Thr Leu Ile 1 5 10 15 Glu Ser Arg Ile Ile Phe Lys Arg Asp Thr Asp Val Asp His His Lys 20 25 30 His Ile Ala Asp Met Tyr Asn Leu Leu Thr Glu Arg Thr Phe Lys Gly 35 40 45 Leu Thr Leu Ala Ile Val Ser Gln Asn Leu Gln Lys Cys Ser Leu Glu 50 55 60 Glu Leu Ser Lys Leu Val Asn Glu Ile Asn Asp Phe Ala Lys Ser Cys 65 70 75 80 Thr Gly Asn Asp Lys Thr Pro Glu Cys Glu Lys Pro Ile Gly Thr Leu 85 90 95 Phe Tyr Asp Lys Leu Cys Ala Asp Pro Lys Val Gly Val Asn Tyr Glu 100 105 110 Trp Ser Lys Glu Cys Cys Ser Lys Gln Asp Pro Glu Arg Ala Gln Cys 115 120 125 Phe Arg Ala His Arg Val Phe Glu His Asn Pro Val Arg Pro Lys Pro 130 135 140 Glu Glu Thr Cys Ala Leu Phe Lys Glu His Pro Asp Asp Leu Leu Ser 145 150 155 160 Ala Phe Ile His Glu Glu Ala Arg Asn His Pro Asp Leu Tyr Pro Pro 165 170 175 Ala Val Leu Leu Leu Thr Gln Gln Tyr Gly Lys Leu Val Glu His Cys 180 185 190 Cys Glu Glu Glu Asp Lys Asp Lys Cys Phe Ala Glu Lys Met Lys Glu 195 200 205 Leu Met Lys His Ser His Ser Ile Glu Asp Lys Gln Lys His Phe Cys 210 215 220 Trp Ile Val Asn Asn Tyr Pro Glu Arg Val Ile Lys Ala Leu Asn Leu 225 230 235 240 Ala Arg Val Ser His Arg Tyr Pro Lys Pro Asp Phe Lys Leu Ala His 245 250 255 Lys Phe Thr Glu Glu Thr Thr His Phe Ile Lys Asp Cys Cys His Gly 260 265 270 Asp Met Phe Glu Cys Met Thr Glu Arg Leu Glu Leu Ser Glu His Thr 275 280 285 Cys Gln His Lys Asp Glu Leu Ser Thr Lys Leu Glu Lys Cys Cys Asn 290 295 300 Leu Pro Leu Leu Glu Arg Thr Tyr Cys Ile Val Thr Leu Glu Asn Asp 305 310 315 320 Asp Val Pro Ala Glu Leu Ser Lys Pro Ile Thr Glu Phe Thr Glu Asp 325 330 335 Pro His Val Cys Glu Lys Tyr Ala Glu Asn Lys Glu Ser Phe Leu Glu 340 345 350 Arg Ile Ser Pro Trp Gln Ser Gln Glu Thr Pro Glu Leu Ser Glu Gln 355 360 365 Phe Leu Leu Gln Ser Ala Lys Glu Tyr Glu Ser Leu Leu Asn Lys Cys 370 375 380 Cys Phe Ser Asp Asn Pro Pro Glu Cys Tyr Lys Asp Gly Ala Asp Arg 385 390 395 400 Phe Met Asn Glu Ala Lys Glu Arg Phe Ala Tyr Leu Lys Gln Asn Cys 405 410 415 Asp Ile Leu His Glu His Gly Glu Tyr Leu Phe Glu Asn Glu Leu Leu 420 425 430 Ile Arg Tyr Thr Lys Lys Met Pro Gln Val Ser Asp Glu Thr Leu Ile 435 440 445 Gly Ile Ala His Gln Met Ala Asp Ile Gly Glu His Cys Cys Ala Val 450 455 460 Pro Glu Asn Gln Arg Met Pro Cys Ala Glu Gly Asp Leu Thr Ile Leu 465 470 475 480 Ile Gly Lys Met Cys Glu Arg Gln Lys Lys Thr Phe Ile Asn Asn His 485 490 495 Val Ala His Cys Cys Thr Asp Ser Tyr Ser Gly Met Arg Ser Cys Phe 500 505 510 Thr Ala Leu Gly Pro Asp Glu Asp Tyr Val Pro Pro Pro Val Thr Asp 515 520 525 Asp Thr Phe His Phe Asp Asp Lys Ile Cys Thr Ala Asn Asp Lys Glu 530 535 540 Lys Gln His Ile Lys Gln Lys Phe Leu Val Lys Leu Ile Lys Val Ser 545 550 555 560 Pro Lys Leu Glu Lys Asn His Ile Asp Glu Trp Leu Leu Glu Phe Leu 565 570 575 Lys Met Val Gln Lys Cys Cys Thr Ala Asp Glu His Gln Pro Cys Phe 580 585 590 Asp Thr Glu Lys Pro Val Leu Ile Glu His Cys Gln Lys Leu His Pro 595 600 605 119572PRTXenopus (Silwana) tropicalis 119Met Asn Ala Leu Met Arg Arg Ala Cys Cys Gly Ala Leu Phe Pro Leu 1 5 10 15 Ser Phe Arg Leu Ala Ala Leu Ser Pro Met Lys Gly Ala Ser Asn Phe 20 25 30 Ser Cys Gly Asn Val Cys Ala Ser Pro Ala Gly Cys Trp Ala Pro Pro 35 40 45 Ser Gly His Asp Thr Gly Ile Lys Val Tyr Asn Ser Leu Thr Arg Arg 50 55 60 Lys Asp Pro Leu Ile Leu Ala Asp Pro Thr Val Ala Thr Trp Tyr Ser 65 70 75 80 Cys Gly Pro Thr Val Tyr Asp His Ala His Leu Gly His Ala Cys Ser 85 90 95 Tyr Val Arg Phe Asp Ile Ile Arg Arg Ile Leu Leu Lys Val Phe Gly 100 105 110 Ile Asp Thr Val Val Val Met Val Val Thr Asp Ile Asp Asp Lys Ile 115 120 125 Ile Lys Arg Ala Lys Glu Leu Asn Ile Ser Pro Val Ala Leu Ala Arg 130 135 140 Thr Tyr Glu Gln Asp Phe Lys Gln Asp Met Thr Ala Leu Lys Val Leu 145 150 155 160 Pro Pro Thr Val Tyr Met Arg Val Thr Glu Asn Ile Pro Gln Ile Ile 165 170 175 Ser Phe Ile Glu His Ile Ile Ala Asn Gly Tyr Ala Tyr Ala Thr Ser 180 185 190 Gln Gly Asn Val Tyr Phe Asp Val Gln Ser Ile Gly Glu Arg Tyr Gly 195 200 205 Lys Phe Asn Asp Ser Phe Ser Asp Thr Ala Ser Glu Ser Ala Ser Gln 210 215 220 Asp Lys Arg His Ile Arg Asp Phe Ala Leu Trp Lys Thr Ser Lys Pro 225 230 235 240 Glu Glu Pro Tyr Trp Ala Ser Pro Trp Gly Lys Gly Arg Pro Gly Trp 245 250 255 His Ile Glu Cys Ser Thr Ile Ala Ser Ser Val Phe Gly Lys His Leu 260 265 270 Asp Ile His Thr Gly Gly Ile Asp Leu Ala Phe Pro His His Glu Asn 275 280 285 Glu Ile Ala Gln Cys Glu Ala Tyr His Gln Ser Thr Gln Trp Gly Asn 290 295 300 Tyr Phe Leu His Thr Gly His Leu His Leu Lys Gly Asn Glu Glu Lys 305 310 315 320 Met Ser Lys Ser Leu Arg Asn Tyr Leu Thr Val Lys Glu Phe Leu Lys 325 330 335 Ser Phe Ser Pro Asp Gln Phe Arg Met Phe Cys Leu Arg Ser Lys Tyr 340 345 350 Lys Ser Ala Val Glu Tyr Ser Asn Gly Ser Met His Asp Ala Val Asn 355 360 365 Thr Leu His Thr Ile Ser Ser Phe Val Asp Asp Ala Lys Ala Tyr Met 370 375 380 Lys Gly Gln Leu Ile Cys Gln Pro Val Gln Glu Ala Leu Leu Trp Gln 385 390 395 400 Arg Leu Asn Glu Thr Lys Val Asn Val Lys Ala Ala Phe Ser Asp Asp 405 410 415 Phe Asp Thr Pro Arg Ala Val Asp Ala Val Met Asp Leu Ile His His 420 425 430 Gly Asn Arg Gln Leu Lys Ala Val Ser Lys Glu Ser Asn Ser Pro Arg 435 440 445 Ser Ser Val Val Tyr Gly Ala Met Ile Ser Tyr Ile Glu Gln Phe Leu 450 455 460 Glu Ile Leu Gly Ile Ser Leu Ser Gln Asn Gln Val Ala Ala Glu Asp 465 470 475 480 Arg His Ser Ala Val Leu Phe Asn Val Val Glu Glu Met Ile Ser Phe 485 490 495 Arg Ser Lys Val Arg Asn Tyr Ala Leu Ala Ala Asp Glu Ser Pro Asn 500 505 510 Ala Ile Gly Gln Glu Glu Lys Gln Gln Tyr Lys Glu Arg Arg Arg Gln 515 520 525 Leu Leu Leu Glu Arg Glu Pro Leu Leu Gln Ala Cys Asp Ile Met Arg 530 535 540 Gln His Leu Ala Val Tyr Gly Ile Asn Val Lys Asp Arg Gly Asn Thr 545 550 555 560 Ser Thr Trp Glu Leu Leu Asp Arg Lys Glu Glu Thr 565 570 120626PRTAmbystoma maculatum 120Met Lys Trp Ala Thr Leu Ile Ser Ile Val Ile Val Leu Ser Cys Thr 1 5 10 15 Glu Ser Arg Ile Leu Asn Lys Arg His His His Glu Gly His Val Asp 20 25 30 Asn Pro Pro His Leu Ile Gly Asp Leu Ile Pro Met Ile Gly Val Asp 35 40 45 Asn Ser Lys Gly Leu Val Leu Ala Ala Val Ser Gln Met Leu Pro Leu 50 55 60 Cys Pro Tyr Glu Glu His Leu Gln Arg Val Glu Asp Val Met Gln Ile 65 70 75 80 Ala Asp Leu Cys Ala Lys Gly Ala Arg His Ala Asn Cys Ala Lys Ser 85 90 95 Pro Met Thr Ile Ile Leu Asp Glu Leu Cys Lys Lys Pro Glu Asn Ala 100 105 110 Glu Lys Tyr Pro Phe His Gln Glu Cys Cys Lys Lys Glu Asp Pro Glu 115 120 125 Arg His Lys Cys Phe Val Glu His Lys Met Ala Asn His Glu Glu Leu 130 135 140 Thr Lys Tyr Val Arg Pro Ala Pro Glu Gln Ile Cys Lys Asp His Ala 145 150 155 160 Glu Asn Arg Gly Pro Leu Leu Ala Arg Tyr Ile Phe Met Leu Ala Ile 165 170 175 Gly His Pro His Met Tyr Ile Pro Ala Ile Leu Gly Phe Ala Gln Arg 180 185 190 Phe Asp Gly Ile Val Ser His Cys Cys Lys Asp Val Glu Thr Ala Gly 195 200 205 Gln Cys Phe Asn Asp Lys Met Pro Glu His Lys Gln Glu Val Glu Tyr 210 215 220 Val Cys Ala Leu Gln Lys His Asn Cys Tyr Ile Leu Gln Asp Phe Lys 225 230 235 240 Glu Arg Ala Leu Thr Ala Tyr Lys Ala Val Gln Ala Ser Gln Lys Phe 245 250 255 Pro Leu Ala Ser Phe Glu Asn Val Gln Ile Ile Val Pro Asp Thr Val 260 265 270 His Leu His Gln Thr Cys Cys Gly Gly Asp Met Met Ala Cys Met Leu 275 280 285 Glu Arg Met Lys Leu Thr Ala Lys Ile Cys Glu Lys Lys Asp Glu Leu 290 295 300 Ala Thr His Leu Lys Glu Cys Cys Asp Lys Pro Leu Leu Glu Arg Ser 305 310 315 320 Ala Cys Ile Ile Arg Leu Pro Asn Asp Gln Lys Pro Ala Asp Leu Ser 325 330 335 Pro Lys Val Pro His Tyr Ile Asp Asp Pro Glu Val Cys Lys Leu Tyr 340 345 350 Thr Glu Gly Gly Asp Thr Phe Met Gly Arg Phe Leu Tyr Glu Cys Ala 355 360 365 Arg Arg His Gln Asp Tyr Ser Pro Glu Met Leu Leu Arg Met Gly Ser 370 375 380 Gly Tyr Glu Glu Phe Leu Lys Lys Cys Cys Ala Ala Glu Gly His Asn 385 390 395 400 Glu Cys Leu Ala Lys Thr Glu Glu Ser Leu Lys Lys Glu Ile Glu Ser 405 410 415 Ser Val Thr Leu Leu Lys Thr Asn Cys Gly Ala Leu Asp Lys Leu Lys 420 425 430 Ser Tyr Leu Phe Gln Asn Leu Leu Ile Phe Lys Tyr Val Ala Arg Met 435 440 445 Pro Ala Leu Ser Glu Gln Ser Leu Leu Arg Ile Thr Lys Ser Met Thr 450 455 460 Thr Ile Gly Glu Lys Cys Cys His Arg Pro Glu Asp Gln Gln Met Thr 465 470 475 480 Cys Ser Glu Gly Gly Leu Gly Ile Val Phe Gly Gln Ile Cys Met Lys 485 490 495 Gln Lys Thr Thr Pro Val Asn Glu Lys Val Ala Gln Cys Cys Ser His 500 505 510 Ser Leu Ser Ser Gln Thr Pro Cys Phe Ser Ala Leu Pro Val Asp Glu 515 520 525 Thr Tyr Val Pro Pro Pro Leu Ser Val Ala Ser Phe Asn Phe Asn Asp 530 535 540 Glu Leu Cys Thr Thr Ser Glu Pro Glu Gln Gln Ser Lys Lys Gln Val 545 550 555 560 Phe Leu Ile Arg Leu Met Lys Gln Tyr Pro His Met Thr Asp Glu Gln 565 570 575 Leu Lys Thr Cys Val Val Asn Phe Val Pro Met Val Asp Gln Cys Cys 580 585 590 Lys Ala Asp Asn His Asn Glu Cys Phe Ala Leu Glu Gly Ala Lys Leu 595 600 605 Ile Asp Ala Cys Lys Ala Ile Leu Ala Val His Pro Ala Val Glu Val 610 615 620 Ser Val 625 121608PRTSalmo salar 121Met Gln Trp Leu Ser Val Cys Ser Leu Leu Val Leu Leu Ser Val Leu 1 5 10 15 Ser Arg Ser Gln Ala Gln Asn Gln Ile Cys Thr Ile Phe Thr Glu Ala 20 25 30 Lys Glu Asp Gly Phe Lys Ser Leu Ile Leu Val Gly Leu Ala Gln Asn 35 40 45 Leu Pro Asp Ser Thr Leu Gly Asp Leu Val Pro Leu Ile Ala Glu Ala 50 55 60 Leu Ala Met Gly Val Lys Cys Cys Ser Asp Thr Pro Pro Glu Asp Cys 65 70 75 80 Glu Arg Asp Val Ala Asp Leu Phe Gln Ser Ala Val Cys Ser Ser Glu 85 90 95 Thr Leu Val Glu Lys Asn Asp Leu Lys Met Cys Cys Glu Lys Thr Ala 100 105 110 Ala Glu Arg Thr His Cys Phe Val Asp His Lys Ala Lys Ile Pro Arg 115 120 125 Asp Leu Ser Leu Lys Ala Glu Leu Pro Ala Ala Asp Gln Cys Glu Asp 130 135 140 Phe Lys Lys Asp His Lys Ala Phe Val Gly Arg Phe Ile Phe Lys Phe 145 150 155 160 Ser Lys Ser Asn Pro Met Leu Pro Pro His Val Val Leu Ala Ile Ala 165 170 175 Lys Gly Tyr Gly Glu Val Leu Thr Thr Cys Cys Gly Glu Ala Glu Ala 180 185 190 Gln Thr Cys Phe Asp Thr Lys Lys Ala Thr Phe Gln His Ala Val Met 195 200 205 Lys Arg Val Ala Glu Leu Arg Ser Leu Cys Ile Val His Lys Lys Tyr 210 215 220 Gly Asp Arg Val Val Lys Ala Lys Lys Leu Val Gln Tyr Ser Gln Lys 225 230 235 240 Met Pro Gln Ala Ser Phe Gln Glu Met Gly Gly Met Val Asp Lys Ile 245 250 255 Val Ala Thr Val Ala Pro Cys Cys Ser Gly Asp Met Val Thr Cys Met 260 265 270 Lys Glu Arg Lys Thr Leu Val Asp Glu Val Cys Ala Asp Glu Ser Val 275 280 285 Leu Ser Arg Ala Ala Gly Leu Ser Ala Cys Cys Lys Glu Asp Ala Val 290 295 300 His Arg Gly Ser Cys Val Glu Ala Met Lys Pro Asp Pro Lys Pro Asp 305 310 315 320 Gly Leu Ser Glu His Tyr Asp Ile His Ala Asp Ile Ala Ala Val Cys 325 330 335 Gln Thr Phe Thr Lys Thr Pro Asp Val Ala Met Gly Lys Leu Val Tyr 340 345 350 Glu Ile Ser Val Arg His Pro Glu Ser Ser Gln Gln Val Ile Leu Arg 355 360 365 Phe Ala Lys Glu Ala Glu Gln Ala Leu Leu Gln Cys Cys Asp Met Glu 370 375 380 Asp His Ala Glu Cys Val Lys Thr Ala Leu Ala Gly Ser Asp Ile Asp 385 390 395 400 Lys Lys Ile Thr Asp Glu Thr Asp Tyr Tyr Lys Lys Met Cys Ala Ala 405 410 415 Glu Ala Ala Val Ser Asp Asp Ser Phe Glu Lys Ser Met Met Val Tyr 420 425 430 Tyr Thr Arg Ile Met Pro Gln Ala Ser Phe Asp Gln Leu His Met Val 435 440 445 Ser Glu Thr Val His Asp

Val Leu His Ala Cys Cys Lys Asp Glu Gln 450 455 460 Gly His Phe Val Leu Pro Cys Ala Glu Glu Lys Leu Thr Asp Ala Ile 465 470 475 480 Asp Ala Thr Cys Asp Asp Tyr Asp Pro Ser Ser Ile Asn Pro His Ile 485 490 495 Ala His Cys Cys Asn Gln Ser Tyr Ser Met Arg Arg His Cys Ile Leu 500 505 510 Ala Ile Gln Pro Asp Thr Glu Phe Thr Pro Pro Glu Leu Asp Ala Ser 515 520 525 Ser Phe His Met Gly Pro Glu Leu Cys Thr Lys Asp Ser Lys Asp Leu 530 535 540 Leu Leu Ser Gly Lys Lys Leu Leu Tyr Gly Val Val Arg His Lys Thr 545 550 555 560 Thr Ile Thr Glu Asp His Leu Lys Thr Ile Ser Thr Lys Tyr His Thr 565 570 575 Met Lys Glu Lys Cys Cys Ala Ala Glu Asp Gln Ala Ala Cys Phe Thr 580 585 590 Glu Glu Ala Pro Lys Leu Val Ser Glu Ser Ala Glu Leu Val Lys Val 595 600 605 122527PRTSphenodon punctatus 122Glu Asp Pro Thr Cys Leu Lys Ser Leu Asp Thr Ile Phe Leu Asp Glu 1 5 10 15 Ile Cys His Glu Glu Gly Phe Ala Ala Lys Tyr Asp Leu Ala Ala Cys 20 25 30 Cys Ala Lys Ala Glu Val Glu Arg Lys Glu Cys Leu Leu Ala His Lys 35 40 45 Asn Ala Thr Pro Gly Phe Ile Pro Ala Phe Gln Arg Pro Gly Ile Glu 50 55 60 Val Ser Cys Lys Leu Tyr Gln Asp Asp Arg Leu Thr Leu Leu Gly Asn 65 70 75 80 Tyr Ile Tyr Glu Val Ala Arg Arg His Pro Tyr Leu Gln Val Pro Pro 85 90 95 Val Phe Ala Thr Ala Ser Leu Tyr Asp Glu Ala Leu Lys Thr Cys Cys 100 105 110 Gln Thr Ala Asp Lys Ala Thr Cys Phe His Pro Arg Ile Pro Pro Leu 115 120 125 Ile Glu Tyr Leu Lys Met Ser Asn Gly Ile Gln Glu Asn Thr Cys Gly 130 135 140 Ile Leu Lys Lys Phe Gly Glu Arg Thr Leu Lys Ala Thr Lys Leu Val 145 150 155 160 Gln Met Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Thr Ile Asn Lys 165 170 175 Leu Val Glu Asp Ile Thr His Met His Thr Glu Cys Cys Arg Gly Asp 180 185 190 Thr Leu Glu Cys Leu Arg Asp Arg Glu Ala Leu Thr Glu Tyr Thr Cys 195 200 205 Ser His Lys Asp Ala Ile Ser Ser Lys Leu Pro Thr Cys Cys Glu Lys 210 215 220 Ser Val Leu Glu Arg Gly Glu Cys Ile Val Arg Leu Glu Asn Asp Asp 225 230 235 240 Lys Pro Ala Asp Leu Ser Glu Arg Ile Ala Glu Tyr Ile Glu Asp Pro 245 250 255 His Val Cys Asp His Leu Ala Lys Glu Gln Asp Ala Phe Leu Ala Lys 260 265 270 Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Leu Ser Thr Gln Ile 275 280 285 Leu Leu Gly Val Gly Lys Gly Tyr Gln Glu Leu Leu Glu Arg Cys Cys 290 295 300 Lys Thr Asp Asn Pro Pro Glu Cys Tyr Gly Gln Ala Glu Ala Asp Leu 305 310 315 320 Lys Lys His Ile Ala Gln Phe Gln Glu Leu Val Gln Gln Asn Cys Asp 325 330 335 Leu Tyr Asn Thr Leu Gly Gly Tyr Leu Phe His Asn Ala Leu Leu Ile 340 345 350 Arg Tyr Thr Lys Arg Met Pro Gln Leu Thr Ser Glu Glu Leu Ile Phe 355 360 365 Tyr Thr Arg Ile Thr Lys Ala Ala Ser Arg Cys Cys Glu Val Ser Val 370 375 380 Asp Lys Lys Leu Pro Cys Thr Glu Gly Tyr Val Asp Phe Val Leu Gly 385 390 395 400 Gln Ile Cys Gln Arg His Gln Arg Ser Ser Ile Asn Val Asn Val Cys 405 410 415 Gln Cys Cys Ser Asn Ser Tyr Ala Leu Arg Ser Leu Cys Ile Thr Ser 420 425 430 Leu Gly Gly Asp Glu Lys Phe Val Pro Ile Glu Phe Ser Ala Asp Leu 435 440 445 Phe Thr Phe His Glu Asp Leu Cys His Ala Ala Gln Asp Lys Leu Gln 450 455 460 Glu Arg Lys Gln Gln Met Ile Val Asn Leu Val Lys His Lys Pro Asn 465 470 475 480 Ile Thr Lys Glu Gln Leu Gln Thr Val Phe Gly Gly Phe Thr Lys Met 485 490 495 Thr Glu Lys Cys Cys Lys Ala Glu Asp His Glu Ala Cys Phe Gly Glu 500 505 510 Glu Gly Pro Lys Leu Val Ala Glu Ser Gln Thr Ala Leu Ala Ala 515 520 525 123101PRTNeoceratodus forsterimisc_feature(79)..(79)Xaa can be any naturally occurring amino acid 123Asp Ala Glu His Lys Ser Asn Ile Cys Lys His Phe Gln Val Val Gly 1 5 10 15 Glu Glu Lys Phe Lys Asn Ile Ile Leu Val Thr Gln Asp Gly His Gly 20 25 30 Pro Phe Ile Gln Val Ser Lys Glu Glu Gln Cys Lys His Tyr Ala Glu 35 40 45 Asn Arg Val Pro Tyr Met Gly Asn Phe Ile Tyr Thr Ala Ala Lys Arg 50 55 60 His Pro Asp Leu Pro Ala Thr Glu Val Leu Ile Tyr Ala Phe Xaa Tyr 65 70 75 80 Glu Ser Gly Ala Val Leu Val Ser Tyr Pro Glu Met Val Gly Cys Cys 85 90 95 Pro Pro Asp Val Leu 100 124614PRTNaja kaouthia 124Met Lys Trp Val Ile Phe Ile Ser Leu Leu Cys Leu Val Ser Phe Ala 1 5 10 15 Glu Val Lys Asn Leu Pro Arg Arg Tyr Arg His Val Asp Asp Gln His 20 25 30 Ser Thr Ile Arg Leu Ala Ser Gln Ile Ser Ala Thr Asp Phe Gly Ala 35 40 45 Ile Thr Leu Thr Leu Val Thr Gln Thr Val Pro Asn Ala Thr Leu Glu 50 55 60 Asp Leu Lys Lys Leu Ser Ala Glu Ile Ile Glu Leu His Lys Lys Cys 65 70 75 80 Val Ala Ser Glu Phe Ser Asp Pro Pro Cys Thr Lys Pro Leu Gly Ile 85 90 95 Val Phe Leu Asp Val Leu Cys His Asn Glu Glu Phe Ser Asn Lys Tyr 100 105 110 Gly Ile Asn Asp Cys Cys Ala Lys Ala Asp Pro Asp Arg Asn Glu Cys 115 120 125 Val Leu Ser His Lys Thr Ser Ser Thr Gly Thr Ile Ser Pro Phe Val 130 135 140 His Pro Asn Ala Glu Glu Ala Cys Gln Ala Phe Gln Asn Asp Arg Asp 145 150 155 160 Ser Val Leu Ala Gln Tyr Ile Phe Glu Leu Ser Arg Arg Tyr Pro Thr 165 170 175 Ala Leu Ser Val Val Ile Leu Glu Ser Thr Lys Thr Tyr Lys Lys Ile 180 185 190 Leu Glu Thr Cys Cys Ala Glu Ala Asp Lys Asp Ala Cys Ile His Glu 195 200 205 Lys Ala Thr Glu Ala Lys Lys Lys Phe Arg Glu Ile Met Glu Glu Gln 210 215 220 Glu Tyr Thr Cys Tyr Asn Leu Lys Lys Tyr Gly Lys Asp Lys Leu Tyr 225 230 235 240 Ala Leu Lys Phe Ile Glu Thr His Glu Lys Phe Val Asn Ala Lys Leu 245 250 255 Glu Thr Ile Thr Gly Ile Ala Glu Phe Val Val His Ile Tyr Glu Glu 260 265 270 Ile Cys Met Gly Asp Ser Val Asp Val Leu Val Asp Arg Ala Ala Leu 275 280 285 Ser Gln Tyr Val Cys Glu His Lys Asp Ala Ile Ser Ser Asn Val Gly 290 295 300 His Cys Cys Glu Lys Pro Leu Val Glu Arg Pro Asn Cys Leu Ala Thr 305 310 315 320 Leu Ala Asn Asp Ala Arg Ser Pro Asp Leu Pro Pro Pro Ser Glu Glu 325 330 335 Ile Leu Lys Glu Thr Glu Ala Cys Thr Thr Tyr Thr Glu Gln Arg Glu 340 345 350 Asn Tyr Lys Glu Ser Phe Leu Phe Thr Leu Thr Arg Asn His Pro Glu 355 360 365 Leu Ser Lys Leu Ile Asp Leu Glu Ile Leu Tyr Lys Tyr Glu Lys Leu 370 375 380 Leu Glu Glu Cys Cys Gln Ser Glu His His Val Gln Cys Leu His Gly 385 390 395 400 Gly Glu Gln Val Phe Lys Leu Tyr Ile Thr Lys Ile Asn Glu Val Val 405 410 415 Lys Ser Asn Cys Asp Ser Tyr Lys Glu Leu Gly Asp Tyr Phe Phe Thr 420 425 430 Asn Glu Phe Leu Val Lys Tyr Ser Arg Met Met Pro Gln Ala Pro Thr 435 440 445 Ser Phe Leu Ile Glu Leu Thr Glu Lys Val Gly Lys Val Ala Glu Lys 450 455 460 Cys Cys Asn Leu Asp Ser Asn His Gln Val Ser Cys Ala Leu Glu Asn 465 470 475 480 Thr Asp Lys Val Met Gly Ser Ile Cys Lys Tyr His Asn Lys His Phe 485 490 495 Ile Asn Asp Gln Ile Cys His Cys Cys Asn Ser Ser Phe Ile Ser Arg 500 505 510 Trp Glu Cys Ile Ser Asn Leu Gly Pro Asp Leu Ser Phe Val Pro Pro 515 520 525 Thr Phe Asn Pro Lys Thr Met Asp Asn Pro Glu Lys Leu Cys Ser Thr 530 535 540 Ser Glu Asp Thr Val Gln Lys Ser Lys Lys Gly Leu Leu Ser Glu Leu 545 550 555 560 Val Lys Ser Lys Pro Asn Ile Ser Glu Glu Glu Leu Ala Ala Thr Ile 565 570 575 Leu Thr Phe Arg Glu Ile Gln Lys Leu Cys Cys Glu Ala Glu Asn Lys 580 585 590 Lys Glu Cys Phe Asp Lys Lys Gly Gln Glu Met Val Glu His Leu Gln 595 600 605 Asn Gly Pro Thr Thr Glu 610 125608PRTSchistosoma mansoni 125Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Asp Ser Ala 1 5 10 15 Phe Ser Arg Gly Leu Phe Arg Arg Asp Ala His Lys Ser Glu Ile Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45 Ile Ala Phe Ser Gln Phe Leu Gln Lys Cys Pro Tyr Glu Glu His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Ala Ile Pro Thr Leu Arg Asp Ser Tyr Gly Glu Leu Ala 100 105 110 Asp Cys Cys Ala Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu Lys 115 120 125 His Lys Asp Asp His Pro Asn Leu Pro Pro Phe Val Arg Pro Asp Ala 130 135 140 Glu Ala Met Cys Thr Ser Phe Gln Glu Asn Ala Val Thr Phe Met Gly 145 150 155 160 His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Ser Ala Ile Met Thr Glu Cys 180 185 190 Cys Gly Glu Ala Asp Lys Ala Ala Cys Ile Thr Pro Lys Leu Asp Ala 195 200 205 Leu Lys Glu Lys Ala Leu Ala Ser Ser Val Asn Gln Arg Leu Lys Cys 210 215 220 Ser Ser Leu Gln Arg Phe Gly Gln Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Met Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Ile Thr 245 250 255 Lys Leu Ala Thr Asp Leu Thr Lys Leu Thr Glu Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285 Cys Glu Asn Gln Ala Ser Ile Ser Ser Lys Leu Gln Ala Cys Cys Asp 290 295 300 Lys Pro Val Leu Lys Lys Ser His Cys Leu Ser Glu Val Glu Asn Asp 305 310 315 320 Asp Leu Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330 335 Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Ala 355 360 365 Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys 370 375 380 Cys Ala Glu Ala Asp Pro Ser Ala Cys Tyr Gly Lys Val Leu Asp Glu 385 390 395 400 Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Ala Asn Cys 405 410 415 Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile 420 425 430 Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Ala Ala Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Val Leu 450 455 460 Pro Glu Ala Gln Arg Leu Pro Cys Val Glu Asp Tyr Ile Ser Ala Ile 465 470 475 480 Leu Asn Arg Val Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Gln 485 490 495 Val Thr Lys Cys Cys Thr Gly Ser Val Val Glu Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Pro Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Ser Leu Pro Glu Lys Glu 530 535 540 Lys Gln Met Lys Lys Gln Ala Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Gly Pro Gln Leu Arg Thr Val Leu Gly Glu Phe Thr 565 570 575 Ala Phe Leu Asp Lys Cys Cys Lys Ala Glu Asp Lys Glu Ala Cys Phe 580 585 590 Ser Glu Asp Gly Pro Lys Leu Val Ala Ser Ser Gln Ala Ala Leu Ala 595 600 605 12614DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 126gagtcagctg aaaa 1412714DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 127gagtccgcgg aaaa 1412814DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 128aaggcttcgt ctgc 1412914DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 129aaggctagct ctgc 1413015DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 130tctgcttgaa tgtgc 1513115DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 131tctgctcgag tgtgc 1513213DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 132gtgggcagca aat 1313313DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 133gtgggatcca aat 1313414DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 134ggaagtcgat gaaa 1413514DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 135ggaagtcgac gaaa 1413639DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 136cgctagcctc gaggtttaaa cgctagcgag ctcggatcc

3913747DNAArtificial SequenceFragment of nucleic acid encoding albumin to introduce restriction site into SEQ ID NO 2 137catggcgatc ggagctccaa atttgcgatc gctcgagcct aggccgg 47138608PRTSalmo salar 138Met Gln Trp Leu Ser Val Cys Ser Leu Leu Val Leu Leu Ser Val Leu 1 5 10 15 Ser Arg Ser Gln Ala Gln Asn Gln Ile Cys Thr Ile Phe Thr Glu Ala 20 25 30 Lys Glu Asp Gly Phe Lys Ser Leu Ile Leu Val Gly Leu Ala Gln Asn 35 40 45 Leu Pro Asp Ser Thr Leu Gly Asp Leu Val Pro Leu Ile Ala Glu Ala 50 55 60 Leu Ala Met Gly Val Lys Cys Cys Ser Asp Thr Pro Pro Glu Asp Cys 65 70 75 80 Glu Arg Asp Val Ala Asp Leu Phe Gln Ser Ala Val Cys Ser Ser Glu 85 90 95 Thr Leu Val Glu Lys Asn Asp Leu Lys Met Cys Cys Glu Lys Thr Ala 100 105 110 Ala Glu Arg Thr His Cys Phe Val Asp His Lys Ala Lys Ile Pro Arg 115 120 125 Asp Leu Ser Leu Lys Ala Glu Leu Pro Ala Ala Asp Gln Cys Glu Asp 130 135 140 Phe Lys Lys Asp His Lys Ala Phe Val Gly Arg Phe Ile Phe Lys Phe 145 150 155 160 Ser Lys Ser Asn Pro Met Leu Pro Pro His Val Val Leu Ala Ile Ala 165 170 175 Lys Gly Tyr Gly Glu Val Leu Thr Thr Cys Cys Gly Glu Ala Glu Ala 180 185 190 Gln Thr Cys Phe Asp Thr Lys Lys Ala Thr Phe Gln His Ala Ile Ala 195 200 205 Lys Arg Val Ala Glu Leu Lys Ser Leu Cys Ile Val His Lys Lys Tyr 210 215 220 Gly Asp Arg Val Val Lys Ala Lys Lys Leu Val Gln Tyr Ser Gln Lys 225 230 235 240 Met Pro Gln Ala Ser Phe Gln Glu Met Ala Gly Met Val Asp Lys Ile 245 250 255 Val Ala Thr Val Ala Pro Cys Cys Ser Gly Asp Met Val Thr Cys Met 260 265 270 Lys Glu Arg Lys Thr Leu Val Asp Glu Val Cys Ala Asp Glu Ser Val 275 280 285 Leu Ser Arg Ala Ala Gly Leu Ser Ala Cys Cys Lys Glu Asp Ala Val 290 295 300 His Arg Gly Ser Cys Val Glu Ala Met Lys Pro Asp Pro Lys Pro Asp 305 310 315 320 Gly Leu Ser Glu His Tyr Asp Val His Ala Asp Ile Ala Ala Val Cys 325 330 335 Gln Thr Phe Thr Lys Thr Pro Asp Val Ala Met Gly Lys Leu Val Tyr 340 345 350 Glu Ile Ser Val Arg His Pro Glu Ser Ser Gln Gln Val Ile Leu Arg 355 360 365 Phe Ala Lys Glu Ala Glu Gln Ala Leu Leu Gln Cys Cys Asp Met Glu 370 375 380 Asp His Ala Glu Cys Val Lys Thr Ala Leu Ala Gly Ser Asp Ile Asp 385 390 395 400 Lys Lys Ile Thr Asp Glu Thr Asp Tyr Tyr Lys Lys Met Cys Ala Ala 405 410 415 Glu Ala Ala Val Ser Asp Asp Asn Phe Glu Lys Ser Met Met Val Tyr 420 425 430 Tyr Thr Arg Ile Met Pro Gln Ala Ser Phe Asp Gln Leu His Met Val 435 440 445 Ser Glu Thr Val His Asp Val Leu His Ala Cys Cys Lys Asp Glu Pro 450 455 460 Gly His Phe Val Leu Pro Cys Ala Glu Glu Lys Leu Thr Asp Ala Ile 465 470 475 480 Asp Ala Thr Cys Asp Asp Tyr Asp Pro Ser Ser Ile Asn Pro His Ile 485 490 495 Ala His Cys Cys Asn Gln Ser Tyr Ser Met Arg Arg His Cys Ile Leu 500 505 510 Ala Ile Gln Pro Asp Thr Glu Phe Thr Pro Pro Glu Leu Asp Ala Ser 515 520 525 Ser Phe His Met Gly Pro Glu Leu Cys Thr Lys Asp Ser Lys Asp Leu 530 535 540 Leu Leu Ser Gly Lys Lys Leu Leu Tyr Gly Val Val Arg His Lys Thr 545 550 555 560 Thr Ile Thr Glu Asp His Leu Lys Thr Ile Ser Thr Lys Tyr His Thr 565 570 575 Met Lys Asp Lys Cys Cys Ala Ala Glu Asp Gln Ala Ala Cys Phe Thr 580 585 590 Glu Glu Ala Pro Lys Leu Val Ser Glu Ser Ala Glu Leu Val Lys Val 595 600 605

Claims

1) A method of preparing a conjugation-competent polypeptide, comprising: a) providing a three-dimensional model comprising at least one instance of an albumin sequence and, optionally, providing an amino acid sequence of that albumin sequence, b) selecting an amino acid residue in the albumin sequence which corresponds to the first, second, third, fourth or fifth residue relative to the N- or C-terminus of the sequence of the model or of the amino acid sequence or which in each instance of the albumin sequence, relating to the three-dimensional model, fulfills the following conditions: i) solvent surface accessibility of at least 80%; ii) B-factor score of at least 30; iii) no polymorphism known to cause thermal instability; c) substituting the selected residue with Cysteine or inserting Cysteine at the N-side or C-side of the selected residue, d) optionally, making additional alterations to the albumin sequence where each alteration is an amino acid deletion, substitution, or insertion, and e) preparing a polypeptide having the resulting amino acid sequence.