Phage Displaying System Expressing Single Chain Antibody

Abstract

Disclosed are nucleic acid libraries for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and for facilitating production of a disulfide-stabilized single chain antibody. Also disclosed are host cell libraries and phage libraries including the nucleic acid libraries. Further disclosed are methods for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and methods for producing a disulfide-stabilized single chain antibody and non-fusion form thereof.

Description

RELATED APPLICATION

[0001] This application claims priority to U.S. provisional application 61/232,819 filed on Aug. 11, 2009. The prior application is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is related to a phage displaying system expressing disulfide-stabilized single chain antibody variable fragments (sc-dsFv).

BACKGROUND OF THE INVENTION

[0003] A single chain variable fragment (scFv) is a single polypeptide chain antibody fragment having a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains. An scFv displayed as a fusion protein N-terminal to the pIII minor capsid protein on filamentous phage surface is one of the most prominent methods in antibody engineering. It was reported that the small size of the scFv enabled superior tissue-penetrating capabilities over whole IgG or Fab fragment, making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (Michnick, S. W., and Sidhu, S. S. (2008) Nat Chem Biol 4(6), 326-329).

[0004] Yet, under practical application conditions, an scFv scaffold tends to form aggregation. The aggregation has much to do with the stability of the two variable domains and the dimeric interface. The instability of the scFv structure also compromises the fidelity in reproducing the antibody gene products on phage surface, causing biases in favor of more stable scFv molecules over the less stable ones, or selecting non-folded structures on phage surfaces but nevertheless binding to an antigen. This structural instability thus impacts negatively on the applications of scFv in biotechnology and medical uses.

[0005] One way to stabilize the scFv scaffold is to engineer a disulfide bond between the two Fv domains, so that the variable domains can be covalently linked with a disulfide bond. Single chain disulfide-stabilized Fv fragment (sc-dsFv) format was constructed in a single polypeptide chain, as in scFv, with a disulfide framework region (Young, N. M. et al., (1995) FEBS Lett 377(2), 135-139; Worn, A., and Pluckthun, A. (1999) Biochemistry 38(27), 8739-8750). The sc-dsFv molecules could be expressed in E. coli, but not be expressed on phage surface or as soluble form secreted by E. coli in a culture medium, mostly due to severely decreased yield because of the introduction of interface cysteines (Worn, A., and Pluckthun, A. (2001) J Mol Biol 305(5), 989-1010).

[0006] Up to now, phage-displayed sc-dsFv libraries and their applications have not been established.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression, for which the expression with conventional signal sequences was not viable. The optimized signal sequences and related discovering methodologies led to the establishment of phage display systems with the sc-dsFv format, enabling the demonstration and comparison of the performance of the sc-dsFv phage display platform with that of the conventional scFv platform.

[0008] Accordingly, in one aspect, the present invention provides a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of expression constructs, each of which includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3' downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

(a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X- .sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3). Each of X.sub.1-X.sub.10 in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

[0009] In another aspect, the invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of host cells each containing an expression construct that includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond; the second nucleotide sequence is located 3' downstream to the first nucleotide; the signal peptide has the amino acid sequence of (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub- .10AAQPAMAHHHHHHGH (SEQ ID NO:1),

(b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.s- ub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3). each of X.sub.1-X.sub.10 in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

[0010] In another aspect, the invention provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library has a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of the phage. The phage library is prepared by the steps of: providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles; the expression construct that includes a first nucleotide sequence encoding a signal peptide, a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, and a third nucleotide sequence encoding a phage envelop protein; the third nucleotide sequence being located 3' downstream to the second nucleotide sequence; the signal peptide has the amino acid sequence of (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X- .sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1),

(b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.s- ub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

[0011] In addition, the invention provides a sc-dsFv phage display platform. According to the invention, a large scale screening for optimal signal sequences was carried out. In one example of the invention, the signal sequences that were effective for phage-displayed sc-dsFv and non-fusion soluble sc-dsFv secretion in E. coli Amber suppressor strain ER2738 were screened to obtain the sequence preference patterns emerged from the optimum signal sequences.

[0012] In still another aspect, the present invention provides an isolated nucleic acid, having a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The signal peptide has the amino acid sequence of

[0013] (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;

[0014] (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.su- b.8X.sub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), in which X.sub.1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

[0015] (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.- 7X.sub.8X.sub.9X.sub.10HHHGH (SEQ ID NO:3), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

[0016] In a further aspect, the present invention provides a host cell containing the nucleic acid described above.

[0017] In a further more aspect, the present invention provides a phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface. The phage is prepared by a method having the steps of: providing the above-described host cell, and culturing the host cell in a medium under conditions allowing expression of the phage.

[0018] In further another aspect, the present invention provides a method for producing a disulfide-stabilized single chain antibody. The method includes the steps of providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody. The expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond; the signal peptide has the amino acid sequence of:

[0019] (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;

[0020] (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.su- b.8X.sub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), in which X.sub.1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V, X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

[0021] (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.- 7X.sub.8X.sub.9X.sub.10HHHGH (SEQ ID NO:3), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

[0022] In addition, the present invention provides a new signal peptide that facilitates production of disulfide-stabilized single chain antibody, and the nucleic acid encoding the signal peptide.

[0023] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

[0025] FIG. 1 is a schema showing the signal sequence in pCANTAB5E and the constructs of DNA libraries to diversify the tentative signal sequence responsible for the expression of the phage-displayed pIII fusion proteins.

[0026] FIG. 2 is a set of diagrams showing the results of the increased binding to VEGF for phage-displayed sc-dsFv signal sequence variants enriched from the three libraries after selection/amplification cycles, including (A) after each round of selection/amplification cycle, the values of the binding of the rescued phage to immobilized VEGF as measured with ELISA. The ELISA signal strengths are shown in the y-axis, as functions of the selection/amplification cycle; and (B) the numbers of output phage particles titered after each round of selection/amplification cycle for each of the three libraries; the output phage titers, as shown in the y-axis, were plotted against the number of the selection/amplification cycles.

[0027] FIG. 3 is a schema showing the DNA construct of the S5 anti-VEGF sc-dsFv as a pIII fusion protein in the pCANTAB5E phagemid.

[0028] FIG. 4 is a diagram showing VEGF-binding strengths of the phage-displayed anti-VEGF sc-dsFv's from various signal sequence variants with or without fXa digestion. Eight variants with maximal fXa digestion resistance from a 96-well ELISA plate containing 96 randomly picked variants were selected from each of the VEGF-binding enriched libraries after the 4.sup.th round of selection/amplification cycle. These variants were cultured and the rescued phages were allowed to bind to immobilized VEGF with (grey histogram) and without (black histogram) the fXa treatment, and the VEGF-binding strengths (y-axis) were measured with ELISA. The error bars were derived from three repeats of the ELISA measurement.

[0029] FIG. 5 is a diagram showing the binding strengths of phage-displayed anti-HAs scFv/sc-dsFv. One of the scFv phages with specific binding ability to H5, 8a, and the other one with broad-spectrum ability to HAs, 12a, were engineered to disulfide-stabilized scFv (ds-scFv) formats; the sc-dsFv construct was different from the scFv construct in the mutations (L:Gly100Cys & H:Gly44Cys). Avl was negative control of an scFv displayed on the phage; and TAA means the phage does not contain any displayed protein; and various HA subtypes were precoated to ELISA wells to determined binding activity, and the error bars were derived from three repeats of the ELISA measurements.

[0030] FIGS. 6A and B are diagrams showing correlations between sc-dsFv folding quality and resistance to fXa digestion. FIG. 6A shows a comparison of the extents (percentages) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4; both of the axes show the ratio (percent) of the ELISA signal for VEGF-binding after the fXa treatment over the ELISA signal for VEGF-binding before the fXa treatment; the y-axis shows the data from secreted sc-dsFv; the x-axis shows the data from phage-displayed sc-dsFv. FIG. 6B shows a comparison of the extents (percentages) of the interface disulfide bond formation (y-axis) with the folding quality (x-axis) of the sc-dsFv from the optimum signal sequence variants from L4. The sc-dsFv folding quality (x-axis) is represented as the sc-dsFv-VEGF binding ELISA signal divided by western blot signal probed with anti-E tag antibody (E/W, VEGF binding signal divided by secreted sc-dsFv quantity), and then the ratio is normalized with that of anti VEGF scFv (fXa+) (CE/CW, VEGF binding signal divided by secreted scFv quantity), that is, the folding quality is quantified with the ratio: (E/W)/(CE/CW); the error bars in each data point indicate the standard deviations from three repeats of the experiment; the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient was shown in each panel.

[0031] FIGS. 7A and B are diagrams showing stability test of soluble sc-dsFv; including FIG. 7A showing the results of the soluble sc-dsFv incubated at 37.degree. C. as the indicated time shown in the x-axis, and the binding capacities estimated with ELISA against VEGF, shown in y-axis; the ELISA signal was normalized against that of the secreted protein kept at 4.degree. C.; and FIG. 7B showing the fXa resistance percentages of the soluble sc-dsFv plotted against the end binding capacities after 12 days of incubation in 37.degree. C.; the error bars in each data point indicate the standard deviations from three repeats of the experiment, and the coefficient of determination R2 and the p-value from Spearman's rank correlation coefficient are shown in the panel.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0033] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0034] Amino acids can be expressed by three letters or one letters. Table 1 lists standard amino acid abbreviations.

TABLE-US-00001 TABLE 1 Standard amino acid abbreviations Amino Acid 3-Letter 1-Letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0035] Very little is known as to why some sc-dsFv constructs could not be expressed on phage surface, and why the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm. The mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasmic membrane could be a key determinant for the folding. It was unexpectedly found in the invention that for the expression of the displayed protein on the phage surface, alternative sequences in the signal peptide region can modulate the expression level and folding quality of the displayed protein. Accordingly, the invention provides a methodology to systematically optimize the signal sequences for phage-displayed protein expression. Based on the optimized signal sequences and the methodologies of the invention, phage display systems with the sc-dsFv format are established.

[0036] According to the present invention, a nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody is provided. The library has a plurality of expression constructs, each of which includes: a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3' downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

(a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X- .sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

[0037] In one embodiment, each of the expression constructs further includes a third nucleotide encoding a phage coat protein, and the third nucleotide sequence being located 3' downstream to the second nucleotide.

[0038] The term "signal peptide" or "signal sequence" used herein refers to a short (i.e. 3-60 amino acids long) peptide chain that directs the transport of a protein. The signal peptide is known to be responsible for the sec system-dependent translocation of the sc-dsFv-pIII fusion from the translation site in cytoplasm across the periplasmic membrane, a critical process for the integration of the displayed protein on the recombinant phage. Considering the vast signal peptide sequence space needed to be explored, the present invention provides biological combinatorial strategies to diversify the signal peptide sequences with synthetic phage display libraries. The variants in the phage libraries were selected and screened for high expression capabilities, so as to identify the key regions of the signal peptide sequences, including the optimal amino acid sequences, positions and types that are effectively responsible for the sc-dsFv expression on phage surface.

[0039] The term "single chain variable fragment" or "scFv" used herein refers to a single polypeptide chain antibody fragment construct encoding a first variable region and a second variable region, with a flexible linkage peptide connecting the two domains. The first and the second variable region can be either a light chain or a heavy chain variable region. The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. One shortcoming of the scFv scaffold is the aggregation tendency of the scFv molecules under physiological and storage conditions. The aggregation mechanism has much to do with the stability of the two variable domains and the dimeric interface. This structural instability has thus impacted negatively on the utilities of scFv, leading to uncertainties to the outcomes of the selected and screened scFv molecules in terms of their potential applications in biomedicine.

[0040] The term "disulfide-stabilized single chain antibody variable fragment" or "sc-dsFv" used herein refers to a single polypeptide chain containing two variable regions capable of forming an interface disulfide bond, where each of the two variable regions may be a heavy chain variable region or a light chain variable region. According to the invention, the sc-dsFv-pIII fusion protein can be prepared by using the optimal signal sequences capable of directing the sc-dsFv expression on phage surface.

[0041] In an embodiment of the invention, the overlapping segments encompassing the complete signal sequence region governing the protein trafficking of the model anti-VEGF sc-dsFv fusion protein were searched with biological combinatorial methodology for sequence preferences leading to effective expression of the sc-dsFv. The engineering platform established for the disulfide-stabilized antibody variable domain fragment as demonstrated could be used to prepare many of scFv molecules in a more stable structure, which could be carried out under harsh conditions, and have longer shelf-life.

[0042] According to one embodiment of the invention, to select signal sequences for effective expression of anti-VEGF sc-dsFv on M13 phage surface, phage display libraries L2, L3 and L4 were constructed to diversify the signal sequence as shown in FIG. 1, where M13pIII-pelB indicated the signal sequence being the wild type signal sequence for pIII in M13 phage genome in connection with pelB peptidase cleavage site. The complexities of the L2, L3 and L4 phage display library were 3.1.times.10.sup.9, 3.7.times.10.sup.9, and 1.5.times.10.sup.9, respectively. These libraries were designed to efficiently diversify the signal peptide sequences on identifying the optimum signal peptides for expression sc-dsFv.

[0043] In one example of the invention, the expression construct is a phagemid. Among the expression constructs, the nucleotide sequence of the signal peptide, sc-dsFv and the phage coat protein could be operatively linked in a random order. In one preferred example of the invention, the second nucleotide sequence encoding sc-dsFv is located 3' downstream to the first nucleotide encoding the signal peptide, and the third nucleotide sequence encoding the phage coat protein is located 3' downstream to the second nucleotide sequence.

[0044] In one embodiment of the invention, a sc-dsFv library, containing more than one billion sc-dsFv variants, is propagated with an E. coli vector of bacterial phage origin following the method as described by McCafferty, J. et al. (Nature 348(6301), 552-554, 1990). The recombinant phages displaying the sc-dsFv variants can be selected or screened for antigen-binding and re-amplified with the host cells, i.e. E. coli.

[0045] Furthermore, the present invention provides a host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of host cells each containing the aforementioned expression constructs.

[0046] The present invention also provides a phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody. The library includes a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of said phage. The phage library is prepared by the steps of: providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles. The expression construct includes (1) a first nucleotide sequence encoding a signal peptide, (2) a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, and (3) a third nucleotide sequence encoding a phage envelop protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence. The signal peptide has the amino acid sequence of

(a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X- .sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) is one of the 20 naturally occurring amino acid residues.

[0047] On the other hand, a sc-dsFv engineering platform is established for preparation of scFv molecules in a more stable structure in the present invention. Accordingly, the present invention provides an isolated nucleic acid that has a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3' downstream to the first nucleotide. The signal peptide has the amino acid sequence of

[0048] (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;

[0049] (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.su- b.8X.sub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), in which X.sub.1 is A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

[0050] (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.- 7X.sub.8X.sub.9X.sub.10HHHGH (SEQ ID NO:3), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

[0051] According to the invention, the nucleic acid further includes a third nucleotide encoding a phage coat protein. The third nucleotide sequence is located 3' downstream to the second nucleotide sequence.

[0052] In one example of the invention, anti-VEGF sc-dsFv phage display platform was developed. As shown in FIG. 1, expression constructs for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody were designed. Each of the three DNA libraries (L2, L3, and L4) contained ten consecutive NNK degenerate codons covering overlapping regions around the signal sequence. N stands for A, G, T, or C, 25% each; K stands for G or T, 50% each. The NNK degenerated codon represents 32 possible triplet combinations, encoding all 20 natural amino acids and an amber stop codon (TAG). Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (see FIG. 1) can all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.

[0053] In order to further identify binding variants, more than 3000 colonies were randomly selected from each of the libraries L2, L3, and L4 after selection/amplification cycles for enrichment of the binding variants. These phage colonies were individually rescued and spotted on nitrocellulose membranes coated with VEGF (100 .mu.g/30 ml). According to the invention, each of the signal peptides having the amino acid sequences of SEQ ID NO: 5-683 as listed in Tables 2, 3 and 4 was obtained and proved to be capable of facilitating the expression of the sc-dsFv on phage surface. After normalization based on the standard phage solution signals in each of the blocks, the phage-displayed scFv expression efficiency for each of the samples was calculated with the following equation:

Ratio = sample ( CV ) sample ( C 0 ) / control ( CV ) control ( C 0 ) ##EQU00001##

[0054] The value of the sample (CV) is the average normalized signal from VEGF-coated membrane; that of the sample (CO) is the averaged normalized signal from the un-coated and un-blocked membrane. Similarly, those of the control (CV) and control (CO) are the averaged normalized signals for the control phage in the same block where the sample signals are measured on corresponding membrane. The ratio derived from the equation was used to rank the efficiency of the sample phage binding to the immobilized VEGF. All the phage samples with measurable binding strengths with the immobilized VEGF were ranked; the signal sequences of the top fifty ranked phage samples are shown and marked with "*" in Tables 2-4.

[0055] Accordingly, new signal peptides that facilitate production of disulfide-stabilized single chain antibody were obtained (see Example 2). In the embodiment of the invention, the signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-683 were proved to facilitate production of disulfide-stabilized single chain antibody. On the other hand, a new isolated nucleic acid encoding the above mentioned signal peptide was provided as well.

[0056] In a preferred embodiment of the invention, the signal peptide selected from the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 471-519 and 566 was obtained and proved to facilitate production of disulfide-stabilized single chain antibody. Accordingly, the preferred isolated nucleic acid encoding each signal peptide as mentioned was also provided.

[0057] In one example of the present invention, the anti-VEGF sc-dsFv was developed by using the signal peptides as identified and obtained by the method of the present invention. In another example, anti-H5 sc-dsFv against influenza virus was developed (see FIG. 5).

[0058] In order to confirm the formation of disulfide bond in the phage-displayed sc-dsFv variants of the present invention, a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains was constructed. As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa-) bound to immobilized VEGF. In contrast, with the fXa treatment, only the anti-VEGF scFv(fXa-) bound to immobilized VEGF. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. The anti-VEGF scFv(fXa-) was quite insensitive to the treatment of fXa, indicating that no other fXa substrate sequences exist in the displayed protein.

[0059] Unexpectedly, it was found in the present invention that each of the signal peptides having the amino acid sequences of SEQ ID NOS: 5-683 as listed in Tables 2-4 enabled the expression and proper folding of the sc-dsFv structure on the phage-displayed platform. In addition, they resulted in secretion of the soluble non-fusion sc-dsFv in culture media.

[0060] Accordingly, the present invention also provides a method for producing a disulfide-stabilized single chain antibody. The method includes providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody. The expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond. The second nucleotide sequence is located 3' downstream to the first nucleotide. The signal peptide has the amino acid sequence of:

[0061] (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10AAQPAMAHHHHHHGH (SEQ ID NO:1), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;

[0062] (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.su- b.8X.sub.9X.sub.10MAHHHHHHGH (SEQ ID NO:2), in which X.sub.1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or

[0063] (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.- 7X.sub.8X.sub.9X.sub.10HHHGH (SEQ ID NO:3), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

[0064] Similar to the aforementioned experiment, the extent (percentage) of the interface disulfide bond formation of the sc-dsFv from the optimum signal sequence variants from L4 were tested. As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from .about.0% up to 40% of the secreted sc-dsFv molecule. As shown in FIG. 6B, the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction.

[0065] In the present invention, a stability test of soluble sc-dsFv was conducted. As shown in FIGS. 7A and 7B, the sc-dsFv antibody fragment scaffold was indeed substantially more stable than the scFv scafold due to the interface disulfide bond in the sc-dsFv constructs.

[0066] According to the invention, the concentration of sc-dsFv antibody produced by the method disclosed herein was unexpectedly high, and stable. Thus, the present invention provides the sc-dsFv at a high concentration sufficient for coating on a solid phase to produce an array for detection or diagnosis without aggregation, different from the prior art where sc-Fv tends to precipitate under the same concentration due to aggregation.

[0067] Accordingly, the present invention provides an array of disulfide-stabilized single chain antibodies produced by the aforementioned method coated on a solid phase. In one example of the invention, the solid phase may be made from silicon, plastic, nylon, glass, ceramic, photoresist or rubber. In one embodiment of the present invention, a microarray test was established using the disulfide-stabilized single chain antibodies produced by the method of the invention, demonstrating that influenza virus could successfully be detected by an array of a serious dilution of anti-H5 sc-dsFv coated on a glass.

[0068] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation.

EXAMPLES

Preparation 1

VEGF Expression and Purification--Human VEGF-121

[0069] Human VEGF-121 (VEGF-A residue 34-135 receptor binding domain) (Fuh, G. et al., (2006) J. Biol. Chem., 281, 6625-6631) was expressed in E. coli as inclusion body. The refolding and purification of VEGF-A were carried out as described in Chang, H. J., et al., (2009) Structure, 17, 620-631.

Preparation 2

Phage Display Libraries with Diversified Signal Sequences N-terminal to the sc-dsFv-pIII Fusion Protein

[0070] Phage display libraries with diversified sequences in the signal peptide region N-terminal to the sc-dsFv-pIII fusion protein were constructed with pCANTAB5E phagemid (GE-Amersham Biosciences) as shown in FIG. 1. Primers encoded with the sequence diversification shown in FIG. 1 were synthesized by IDT (Integrated DNA Technologies).

[0071] For each of the phage display libraries, phagemid templates were constructed with TAA stop codons inserted in the sequence region for diversification (Huang et al., (2010) J. Biol. Chem., in press). The M13pIII-pelB signal sequence for phage-displayed pIII-fusion protein is a combination of the wild-type M13 signal peptide N-terminal to gene III (MKKLLFAIPLVVPFYSHS) (SEQ ID NO: 684) and the pelB signal sequence of Pectobacterium wasabiae (MKYLLPTAAAGLLLLAAQPAMA) (SEQ ID NO: 685). This merged signal sequence (shown in bold font above) was considered containing the tentative n- h- and c-regions of the signal sequence. DNA libraries were constructed to diversify the amino acid sequence in the key regions. Each of the four of DNA libraries (L2, L3, L7) contained ten consecutive NNK (N stands for 25% of G, C, A, and T, and K stands for 50% of G and T; underlined by dashed lines) degenerate codons covering a portion of the tentative signal sequence. Also shown in the Figure are the sequences containing TAA stop codons (underlined regions) used as the templates for the library constructions. The oligonucleotide-directed mutagenesis procedure initially proposed by Kunkel (Kunkel et al., (1987) Methods Enzymol, 154, 367-382) was used for the phagemid library construction. The TAA stop codons in the phagemid templates ensure that the un-mutated phagemid templates after the mutagenesis procedure are incapable of producing pIII fusion protein for phage surface display (Sidhu and Weiss, (2004) Constructing phage display libraries by oligonucleotide-directed mutagenesis. In: Clackson, T., and Lowman, H. B. (eds). Phage Display, 1st Ed., Oxford University Press, New York).

[0072] After the oligonucleotide-directed mutagenesis procedure, E. coli strand ER2738 was transformed with the phagemid libraries and the recombinant phage particles were rescued with helper phage M13KO7 (GE-Amersham Biosciences). The phage particles were precipitated with PEG/NaCl, and resuspended in PBS. More details of the phage library preparation can be found in a previous publication (Hsu, H. J. et al., (2008) J Biol Chem 283(18), 12343-12353).

[0073] Seven sc-dsFv variants were constructed on the basis of the phagemid encoding the template anti-VEGF scFv(fXa+): S1(L:Gln38Cys & H:Gln39Cys); S2(L:Gly41Cys & H:Gly42Cys); S3(L:Ala43Cys & H:Gln112Cys); S4(L:Phe98Cys & H:Leu45Cys); S5(L:Gln100Cys & H:Gly44Cys); S6(L:Gln38Cys & H:Leu45Cys); S7(L:Ala43Cys & H:Gln112Cys & L:Gln100Cys & H:Gly44Cys). These cysteine pairs were determined by distance constrain for possible disulfide bonds in the model structure (PDB code: 2FJG).

Preparation 3

Biopanning Against VEGF with Phage-Displayed Anti-VEGF sc-dsFv Libraries

[0074] Maxisorb Immune Tubes (Nunc) were coated with VEGF (25 .mu.g in 1 ml PBS in each tube) at 4.degree. C. overnight. The tubes were blocked with 4 ml of 5% skim milk in PBST (PBS with 0.05% Tween 20) for one hour at room temperature with gentle shaking and then washed with PBST. In each of the tubes, 10.sup.11 colony-forming units (cfu) of phage from each of the phage display libraries were mixed with 1 ml of 5% skim milk. The phage particles were allowed to bind to the immobilized VEGF in the tube at room temperature for two hours under gentle shaking. After the binding, the tubes were washed 10 times with PBST and 2 times with PBS. One milliliter of E. coli strand ER2738 in the log phase was added to each of the tubes at room temperature with gentle shaking for 15 minutes. From each tube, the infected E. coli was transferred to 10 ml of a 2YT medium containing 20 .mu.g/ml of ampicillin and was titered with 2YT agar plates containing 100 .mu.g/ml of ampicillin. The infected E. coli was incubated at 37.degree. C. for one hour with vigorous shaking Ampicillin was then added to reach final concentration of 100 .mu.g/ml. The culture was incubated for another hour at 37.degree. C. before transferred to final 100 ml 2YT medium (100 .mu.g/ml of ampicillin) containing 10.sup.11 cfu M13KO7 helper phage. After two hours of incubation, kanamycin was added to final concentration of 70 .mu.g/ml. The culture was incubated at 37.degree. C. overnight with vigorous shaking. The phage in the supernatant of the culture was harvested by centrifugation. The phage was titered, precipitated with PEG/NaCl, and resuspended in PBS. The phage solution was ready for the next round of selection.

Preparation 4

Enzyme-Linked Immunosorbent Assay (ELISA) for Phage-Displayed Anti-VEGF sc-dsFv Binding Against Immobilized VEGF and Anti-E-Tag Antibody

[0075] Single E. coli colonies harboring the selected phagemids were randomly picked using a GENETIX Qpix II colony picker to 96-well deep well culture plates. Each well contained 960 .mu.l 2YT (100 .mu.g/ml of ampicillin and 10 .mu.g/ml of tetracyclin). The culture plates were incubated at 37.degree. C. shaking vigorously for 4 hours before adding 20 .mu.l of M13KO7 helper phage (10'' cfu/ml). The plates were then incubated at 37.degree. C. for one hour with vigorous shaking before adding 20 .mu.l of kanamycin to the final concentration of 50 .mu.g/ml. After overnight incubation at 37.degree. C. with vigorous shaking, the cultures were centrifuged at 3000 g for 10 minutes at 4.degree. C. From each well of the culture plates, 100 .mu.l of the supernatant was mixed with 100 .mu.l of 5% skim milk. Half of the phage mixture was added to a corresponding well of a 96-well Maxisorb microtiter plate precoated with VEGF (1 .mu.g/well) and blocked with 5% skim milk; the other half was added to a corresponding well of another microtiter plate precoated with polyclonal goat anti-E-tag antibody (1 .mu.g/well, Novus Biologicals). After one hour incubation at room temperature, the ELISA plates were washed six times with PBST. The phage particles remained on the plates were measured with HRP-labeled mouse anti-M13 antibody (1/3000, GE Healthcare) and TMB substrate (KPL). The reaction was stopped with 50 .mu.l of 1 N HCl and the signal intensity was measured at OD 450 nm.

Preparation 5

Measurement of Interface Disulfide Bond Formation in Phage-Displayed Anti-VEGF sc-dsFv

[0076] Fifty microliters of a freshly prepared phage supernatant (see above) was mixed with 50 .mu.l of a two-fold concentrated reaction buffer containing 1 unit of bovine factor Xa (fXa) (Novagen) in a Maxisorb microtiter plate precoated with VEGF (1 .mu.g/well) and blocked with 5% skim milk. After two hours of enzymatic reaction at 37.degree. C., the phage particles remained bound to the microtiter plate were measured following the same ELISA procedure as described above.

Preparation 6

Western Blot Assay for the Phage-Displayed Anti-VEGF sc-dsFv

[0077] Single colony phage was amplified, harvested, precipitated with PEG/NaCl, and resuspended in PBS (see above). Phage particles (10.sup.11 cfu) were prepared under either a non-reducing or reducing condition before electrophoresis in a 10% SDS-polyacrylamide gel. After the electrophoresis, the proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated with a monoclonal mouse anti-pIII antibody (1/3000 mg/ml, New England Biolabs) for one hour at room temperature. After three washes (5 minutes each) with PBST, the membrane was incubated with HRP-labeled anti-mouse antibody (1/3000, GE Healthcare) for 1 hour at room temperature. After three washes with 10 ml PBST, the membrane was developed with 4-chloro-1-naphthol (4CN) substrate (KPL) until the desired color intensity was achieved.

Preparation 7

Preparation of Non-Fusion Soluble scFv/sc-dsFv

[0078] Seven hundred and fifty microliters of mid-log phase (OD.sub.600nm=0.6) E. coli host (non-suppressor strain HB2151 or suppressor strain ER2738) grown in a 2YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0) was infected with 50 .mu.l of a phage solution (10.sup.11 cfu/ml). After one hour incubation at 37.degree. C. with shaking, 100 .mu.l ampicillin in a 2YT medium was added to the final concentration of 100 .mu.g/ml. After another hour of incubation, 100 .mu.l isopropyl-beta-D-thiogalactopyranoside (IPTG) in a 2YT medium was added to the final concentration of 1 mM. The culture was kept at 37.degree. C. with vigorous shaking overnight. The secreted soluble scFv or sc-dsFv in the supernatant was separated from the bacterial host by centrifugation at 3000 g for 10 minutes.

Preparation 8

ELISA for Immobilized VEGF Binding

[0079] For phage ELISA, each well in a Maxisorb 96-well microtiter plate (Nunc) was coated with 2 .mu.g VEGF at 4.degree. C. overnight. The wells were blocked with 5% skim milk in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4, 0.1% tween20, pH 7.4) for one hour. After 3.times.300 .mu.l PBST and 2.times.300 .mu.l PBS washes, 100 .mu.l of a phage solution and 100 .mu.l of 5% skim milk in PBST were added to each well and incubated at room temperature with shaking for one hour. After washing each of the wells three times with 300 .mu.l of PBST each and twice with 300 .mu.l of PBS each, the bound phages were labeled with anti-M13 antibody conjugated with HRP (GE-Amersham) 1/3000 dilution in 5% skim milk in PBST for one hour. The ELISA signal was developed by incubating each well with 100 .mu.l of a TMB solution (KPL Inc.) for 5 minutes. The reaction was stopped with 100 .mu.l 1 N HCl, and the optical density was recorded with VICTOR3 Multilabel Plate Readers (Perkin Elmer) at 450 nm.

[0080] For scFv or sc-dsFv ELISA, 100 .mu.l of a soluble scFv solution was used instead of phage solution, and HRP-conjugated protein L (0.5 .mu.g/ml in 5% skim milk in PBS, from Pierce) was used instead of HRP-conjugated anti-M13 antibody. When needed, the ELISA signals were normalized with the signals of the control anti-VEGF scFv in serial dilution.

Preparation 9

fXa Protease Digestion

[0081] For phage solutions, 20 .mu.l (1 unit) of bovine factor Xa (fXa) protease (Novagen) in a six-fold concentrated reaction buffer was added to 100 .mu.l of a phage solution at 37.degree. C. After 2 hours of enzymatic reaction, 100 .mu.l of 5% skim milk in PBST was added to the reaction mixture before the VEGF-binding ELISA measurement was carried out in the manner described in the previous section. The fXa resistance percentage was calculated with the ratio of the ELISA reading in the presence of fXa over the ELISA reading in the absence of fXa. The ELISA readings for the ratio were adjusted by shift the baseline determined with the null control ELISA readings. For soluble scFv/sc-dsFv fXa digestion, all procedures were the same except that the enzymatic reaction was carried out for one hour at room temperature.

Preparation 10

Construction of anti-H5 sc-dsFv against Influenza Virus

[0082] The construction of scFv library derived from mouse spleen after immunization of hemagglutinin from influenza virus was based on the protocols described in "Phage Display, A Laboratory Manual, edited by Carlos F. Barbas Ill., Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA". In brief, after hemagglutinin immunization, the total RNA derived from mouse spleen was purified by Trizol reagent (Invitrogen) according to the manufacturer's protocols. After cDNA synthesis by reverse transcriptase, the gene fragments encoded heavy and light chains of antibody variable region were amplified by the specific primer sets described in the book mentioned above, respectively. The scFv fragments were synthesized by two-steps PCR reactions, and then cloned into a pCANTAB 5E phagemid vector with the signal sequence derived from the library 2 for sc-dsFv phage production. The library complexity was 4.5.times.10.sup.7. After panning against H5, two clones were selected for mono-spectral binding to H5 (clone 8a) and broad spectral binding to H1, H3 and H5 (clone 12a). These two clones were subjected to disulfide bond formation mutants between L100 and H44 (based on Kabat numbering) and then for sc-dsFv phage production and ELISA detection (8aS5 and 12aS5, respectively).

Preparation 11

Microarray Test for sc-dsFv Binding of H5 Influenza Virus

[0083] The just-described sc-dsFv ds-8a or and ds-12a was subcloned into a pET32a vector with thioredoxin as a fusion protein partner at the N-terminus. The thioredoxin-sc-dsFv fusion proteins could be expressed in Rosetta-gami B strain of E. coli in a soluble form. After purification and TEV protease digestion to remove thioredoxin, the purified ds-8a protein was found to have a binding affinity and specificity similar to those of H5 based on an ELISA assay and array studies. The protein ds-AV1, ds-12a and ds-8a were spot on glass slides coated with streptavidin as the purified ds-AV1, ds-12a and ds-8a proteins contained a biotinylated Avitag sequence at their C-termini. The highest protein concentration used for this protein array was 8 mg/ml, 8 mg/ml, and 0.8 mg/ml, respectively. These proteins were 2 fold dilution with a 100 mM sodium phosphate buffer, pH 8.5 from the highest protein concentration for 15 times for spotting (10 nl/spot), and then the protein of each concentration was spotted for 5 replicates. After spotting, each glass slide was sealed to form 16 distinct squares for reaction. After blocking with 5% BSA for 30 minutes, the H5 influenza virus (about .about.10.sup.7 PFU/ml) was added to react with spotted sc-dsFvs for 30 minutes. After 3 times wash with a phosphate buffer for 5 minutes each, 40 nm fluorescence beads coated with ds-8a (.about.10.sup.7) were added to each square and incubated for 30 minutes. After 3 times wash again with a phosphate buffer for 5 minutes each, the glass array was air-dried for detection.

Example 1

Selection of Signal Sequences for Effective Expression of anti-VEGF sc-dsFv on M13 Phage Surface

[0084] Phage display libraries L2, L3, and L4 were constructed to diversify the signal sequence of the S5 anti-VEGF sc-dsFv-pIII fusion protein as shown in FIG. 1. The complete DNA construct and the amino acid sequence of the S5 anti-VEGF sc-dsFv are shown in FIG. 1. The S5 sequence remained unchanged in all the variants from the libraries. The complexities of the L2, L3, and L4 phage display libraries were 3.1.times.10.sup.9, 3.7.times.10.sup.9, and 1.5.times.10.sup.9, respectively. These libraries were designed to diversify the signal peptide sequences in the h-region, c-region, and a few N-terminal residues of the mature phage-displayed anti-VEGF sc-dsFv.

[0085] Each of the phage display libraries was selected for binding against immobilized VEGF. The trends of enrichment of the VEGF-binding phage variants from each of the three libraries, plotted as functions of the number of selection/amplification cycle, are shown in FIG. 2. After four rounds of selection/amplification cycle, the VEGF-binding phage variants were enriched for more than one order of magnitude. The enrichment trends were similar among the variants from the three libraries. This result indicates that the signal sequence regions covered by the three signal sequence libraries (FIG. 1) could all be optimized to increase the expression of the correctly folded anti-VEGF sc-dsFv on phage surface.

Example 2

Interface Disulfide Bond Formation in anti-VEGF sc-dsFv on M13 Phage Surface

[0086] In order to test the formation of the disulfide bond in the phage-displayed sc-dsFv variants, we constructed two control phage-displayed anti-VEGF scFv variants: one with a factor Xa cutting site, -IEGR-, encoded in the linker peptide connecting the two variable domains (anti-VEGF scFv(fXa+)); the other without this fXa cutting site (anti-VEGF scFv(fXa-)). As shown in FIG. 3, the S5 anti-VEGF sc-dsFv was constructed with a fXa substrate sequence (-IEGR-) in the linker sequence between the two variable domains. The cleavage of the fXa substrate sequence in the phage-displayed anti-VEGF scFv(fXa+) resulted in separation of the variable domains, which in turn abolished the affinity of the phage-displayed scFv against immobilized VEGF. Both phage-displayed scFv's did not have the engineered interface disulfide bond as in S5; the scFv(fXa+) construct had the -IEGR- site in the linker peptide (-(G).sub.4SIEGRS(G).sub.4S--), while the scFv(fXa-) construct had the conventional -(G).sub.4S(G).sub.4S(G).sub.4S-- linker peptide.

[0087] As shown in FIG. 4, without the fXa treatment, both anti-VEGF scFv(fXa+) and scFv(fXa-) bound to immobilized VEGF. But with the fXa treatment, only the anti-VEGF scFv(fXa-) bound to immobilized VEGF. In contrast, all the S5 signal sequence variants for the phage-displayed sc-dsFv showed substantial increase in resistance to fXa protease activity, indicating that the interface disulfide bonds in the anti-VEGF sc-dsFv's were formed to stabilize the functional dimeric structure after the cleavage of the peptide linker between the two variable domains. The results unambiguously demonstrated that the engineered interface disulfide bond was correctly formed in the phage-displayed S5 anti-VEGF sc-dsFv from some of the signal sequence variants from all three VEGF-binding enriched signal sequence libraries (L2, L3 and L4).

Example 3

Preference Sequence Patterns of the Optimum Signal Peptides in Effective Expression of Functional Anti-VEGF sc-dsFv

[0088] The functionality of the anti-VEGF sc-dsFv on phage surface was quantified with two quantitative measurements: the affinity of the sc-dsFv against VEGF and the extent of the interface disulfide bond formation. After the tests, 250, 126, and 213 optimum signal sequences were found in L2, L3, and L4 library, respectively, which are summarized as following Tables 2-4. Among them, fifty signal sequence variants with the highest sc-dsFv-VEGF binding affinities selected from more than 3000 random single colonies of the enriched libraries L2, L3, and L4 were marked with the symbol "*." The symbol "q" indicated that the nucleotide sequence TAG (amber stop codon) that could be translated to Gln (Q) with 0.8.about.20% in E. coli amber suppressor strains which were normally used in phage production.

TABLE-US-00002 TABLE 2 Preference sequence patterns selected from L2 S5 sc-dsFv library SEQ ID No. Code Sequence NO: M13-pe1B VKKLL FAIPLVVPFY AAQPAMAHHHHHH 4 1 1.12B VKKLL VLSHLPFMTD AAQPAMAHHHHHH * 5 2 9.26.10B VKKLL SHWLLSSqLQ AAQPAMAHHHHHH * 6 3 2.12A VKKLL AMSLAPSVFP AAQPAMAHHHHHH * 7 4 9.12A VKKLL WSLFFqqLNP AAQPAMAHHHHHH * 8 5 2.12F VKKLL LLSLLQRPLP AAQPAMAHHHHHH * 9 6 1.2H VKKLL LSSWLMTRFP AAQPAMAHHHHHH * 10 7 6.9G VKKLL VLSHFPAFVP AAQPAMAHHHHHH * 11 8 1.8F VKKLL PLLSLPLPPN AAQPAMAHHHHHH * 12 9 7.1B VKKLL VLTPMHFSSP AAQPAMAHHHHHH * 13 10 9.26.10A VKKLL ILALPQSYPL AAQPAMAHHHHHH * 14 11 5.4A VKKLL qALYFSLPSS AAQPAMAHHHHHH * 15 12 YJ2.2 VKKLL VSAMTSASFP AAQPAMAHHHHHH * 16 13 5.2F VKKLL LPASWLFGQP AAQPAMAHHHHHH 17 14 10.2D VKKLL WSLFFqqLNP AAQPAMAHHHHHH * 18 15 YJ2.34 VKKLL FVMALRSSAP AAQPAMAHHHHHH * 19 16 3.3F VKKLL FLWPFYNGHI AAQPAMAHHHHHH 20 17 4.1A VKKLL QSFYLSLqLD AAQPAMAHHHHHH * 21 18 10.7H VKKLL SLTFPFTIHS AAQPAMAHHHHHH * 22 19 1.9D VKKLL WPVLSPSLFP AAQPAMAHHHHHH * 23 20 5.12D VKKLL PWLFSTFPSS AAQPAMAHHHHHH * 24 21 1.8D VKKLL IMSSLPTLSP AAQPAMAHHHHHH * 25 22 4.11F VKKLL IMSRVLAPDF AAQPAMAHHHHHH * 26 23 1.7C VKKLL FDFWFSSFLq AAQPAMAHHHHHH * 27 24 4.8G VKKLL YGqLMLLSSD AAQPAMAHHHHHH * 28 25 4.4E VKKLL PWLFPFHAYP AAQPAMAHHHHHH * 29 26 1.12G VKKLL LVMTLSRQPF AAQPAMAHHHHHH 30 27 4.8A VKKLL ASAYLYHGLS AAQPAMAHHHHHH * 31 28 4.4C VKKLL PFFAGVLqHP AAQPAMAHHHHHH * 32 29 3.11A VKKLL ALSSPFFHIP AAQPAMAHHHHHH * 33 30 10.3F VKKLL PTRqPMMYPP AAQPAMAHHHHHH * 34 31 YJ2.15 VKKLL QLLMPFLNSP AAQPAMAHHHHHH * 35 32 9.9H VKKLL CSLGYACIPP AAQPAMAHHHHHH * 36 33 4.9C VKKLL LMPWLFNSPP AAQPAMAHHHHHH 37 34 3.12B VKKLL LDqLAYAALS AAQPAMAHHHHHH * 38 35 4.10G VKKLL qSTVFFSWLS AAQPAMAHHHHHH * 39 36 YJ2.18 VKKLL LPWALSHQVL AAQPAMAHHHHHH * 40 37 7.2E-q VKKLL ALTYPAFLYD AAQPAMAHHHHHH * 41 38 1.11A VKKLL AMAPPMMSMN AAQPAMAHHHHHH * 42 39 5.3D VKKLL WWSSLFAPSP AAQPAMAHHHHHH 43 40 4.6H VKKLL GSFILARSMD AAQPAMAHHHHHH 44 41 5.11C VKKLL MVLTSWHPYP AAQPAMAHHHHHH * 45 42 2.8C VKKLL FSLRFFFPSS AAQPAMAHHHHHH 46 43 2.5F VKKLL WLWSTPLFPH AAQPAMAHHHHHH 47 44 2.2A VKKLL PLLFSLDGDP AAQPAMAHHHHHH * 48 45 3.2C-d VKKLL SVSLSSYSFY AAQPAMAHHHHHH * 49 46 3.1H VKKLL LNGTFSAqLF AAQPAMAHHHHHH * 50 47 6.4A VKKLL WHVLPYLPNS AAQPAMAHHHHHH * 51 48 4.10E VKKLL SIVPLFSPqS AAQPAMAHHHHHH * 52 49 7.4H VKKLL VMTSPMLAPG AAQPAMAHHHHHH * 53 50 2.5H VKKLL VLSLPSIAPH AAQPAMAHHHHHH 54 51 6.4E VKKLL qSLLLLRALL AAQPAMAHHHHHH * 55 52 2.1A VKKLL FSLPVFFDLP AAQPAMAHHHHHH 56 53 4.11D VKKLL LLFSMARPLP AAQPAMAHHHHHH * 57 54 7.10A VKKLL TqAVFPFTFN AAQPAMAHHHHHH * 58 55 3.2E VKKLL LASWLFRADM AAQPAMAHHHHHH * 59 56 5.2E VKKLL PFLFPFPSPS AAQPAMAHHHHHH * 60 57 YJ2.128 VKKLL ALSAWSLSQT AAQPAMAHHHHHH * 61 58 4.7H VKKLL ALLPLFPTqH AAQPAMAHHHHHH * 62 59 2.10F VKKLL AALASFPPAP AAQPAMAHHHHHH * 63 60 YJ2.22 VKKLL LLMPFLNQSP AAQPAMAHHHHHH * 64 61 7.5A VKKLL FTSGLKLVPP AAQPAMAHHHHHH 65 62 6.10F VKKLL LqPLLSIYLN AAQPAMAHHHHHH 66 63 4.11B VKKLL LSSLWSAYMD AAQPAMAHHHHHH 67 64 2.5C VKKLL LLGqSLMHFQ AAQPAMAHHHHHH 68 65 YJ2.25 VKKLL PQLAMSLPSI AAQPAMAHHHHHH 69 66 10.3H VKKLL YETMLSSYLY AAQPAMAHHHHHH 70 67 3.10D VKKLL SLYYFPLVPY AAQPAMAHHHHHH 71 68 4.7C VKKLL qRTVAAAYFW AAQPAMAHHHHHH 72 69 4.12D VKKLL FLTWLRYGFP AAQPAMAHHHHHH 73 70 6.1A VKKLL LLLTLMqPTS AAQPAMAHHHHHH 74 71 8.10C VKKLL FDFFTHVHLF AAQPAMAHHHHHH 75 72 5.6E VKKLL ALYPHFVSFT AAQPAMAHHHHHH 76 73 4.11E VKKLL LPYAIqLFSP AAQPAMAHHHHHH 77 74 YJ2.5 VKKLL WFPLHSSLLP AAQPAMAHHHHHH 78 75 4.7A VKKLL PALLLATAAF AAQPAMAHHHHHH 79 76 3.11C VKKLL LASVAWNLDS AAQPAMAHHHHHH 80 77 YJ2.121 VKKLL VGSLLFWPQQ AAQPAMAHHHHHH 81 78 4.5F VKKLL SPLLFLqNYT AAQPAMAHHHHHH 82 79 3.2F VKKLL SYWLDFIqVL AAQPAMAHHHHHH 83 80 10.3C VKKLL VPSFLLSPSP AAQPAMAHHHHHH 84 81 9.23.7H VKKLL SLYWLTSqPL AAQPAMAHHHHHH 85 82 3.9A VKKLL FALSSVHSPP AAQPAMAHHHHHH 86 83 4.11H VKKLL SYYSLLYSYP AAQPAMAHHHHHH 87 84 3.1C VKKLL LVSGLqPWYF AAQPAMAHHHHHH 88 85 2.5A VKKLL VLATPLHLSP AAQPAMAHHHHHH 89 86 10.6H-q VKKLL SLAFPLFTPP AAQPAMAHHHHHH 90 87 3.6A VKKLL SLVPIFPFST AAQPAMAHHHHHH 91 88 8.10D VKKLL qPVLFSFFIR AAQPAMAHHHHHH 92 89 4.3B VKKLL MSqFLNLLSP AAQPAMAHHHHHH 93 90 2.3G VKKLL WAVqPLFPLN AAQPAMAHHHHHH 94 91 5.3H VKKLL MFSLVPSPPI AAQPAMAHHHHHH 95 92 10.7B VKKLL PFFLQPFqFP AAQPAMAHHHHHH 96 93 7.2D-q VKKLL PDLLASVLPV AAQPAMAHHHHHH 97 94 2.9H VKKLL FWqFLWPSLP AAQPAMAHHHHHH 98 95 6.4A VKKLL LLGqFFPNPM AAQPAMAHHHHHH 99 96 6.4D VKKLL TLSALSQWHP AAQPAMAHHHHHH 100 97 9.4D VKKLL SLVYFFPFYP AAQPAMAHHHHHH 101 98 10.2H VKKLL FAFAPAPFYH AAQPAMAHHHHHH 102 99 4.12B VKKLL FLPFALVPRQ AAQPAMAHHHHHH 103 100 4.1F VKKLL ALWMqLYPQD AAQPAMAHHHHHH 104 101 YJ2.27 VKKLL ASILFSHAAP AAQPAMAHHHHHH 105 102 2.2C VKKLL LPLPWSLHLY AAQPAMAHHHHHH 106 103 4.9C VKKLL LPHFMSFWFE AAQPAMAHHHHHH 107 104 7.3E VKKLL LFQPFWPIPY AAQPAMAHHHHHH 108 105 4.7F VKKLL LLFSLGRLPP AAQPAMAHHHHHH 109 106 7.12G VKKLL PLWVLLKDPL AAQPAMAHHHHHH 110 107 9.3B VKKLL MSFATLFPHN AAQPAMAHHHHHH 111 108 4.5B VKKLL qHSLVTSWLC AAQPAMAHHHHHH 112 109 5.2H VKKLL LLFqGAFVGq AAQPAMAHHHHHH 113 110 4.4C VKKLL WMFHSLPFSP AAQPAMAHHHHHH 114 111 6.8G VKKLL LTqLLLTRLH AAQPAMAHHHHHH 115 112 4.10A VKKLL ALTLVPSSYP AAQPAMAHHHHHH 116 113 4.5D VKKLL LPWYMLLSDS AAQPAMAHHHHHH 117 114 9.3E VKKLL VVTqFWPSLP AAQPAMAHHHHHH 118 115 4.3G VKKLL LSTLFLWHVR AAQPAMAHHHHHH 119 116 9.7E VKKLL RSLFFqqLYP AAQPAMAHHHHHH 120 117 YJ2.30 VKKLL TLTTLHQTFP AAQPAMAHHHHHH 121 118 1.3B VKKLL SALLAPWYWD AAQPAMAHHHHHH 122 119 8.9B VKKLL AIqqRMQIYT AAQPAMAHHHHHH 123 120 3.4E VKKLL LLFPWFQPPY AAQPAMAHHHHHH 124

121 9.23.7E VKKLL YFTSLLGqFP AAQPAMAHHHHHH 125 122 6.3D VKKLL PVLIFLSEIR AAQPAMAHHHHHH 126 123 9.5G VKKLL VATSLRWAVT AAQPAMAHHHHHH 127 124 YJ2.54 VKKLL AQLFHLFATH AAQPAMAHHHHHH 128 125 8.6G VKKLL LqFSALFNSF AAQPAMAHHHHHH 129 126 7.12C-q VKKLL FHLMSMLPPP AAQPAMAHHHHHH 130 127 5.4C VKKLL PVCSqSMFPI AAQPAMAHHHHHH 131 128 YJ2.48 VKKLL LLLSSSYQSP AAQPAMAHHHHHH 132 129 4.3D VKKLL LDSLFFHAPL AAQPAMAHHHHHH 133 130 7.7A VKKLL qAWVFSAHQL AAQPAMAHHHHHH 134 131 YJ2.99 VKKLL FQALGALTSP AAQPAMAHHHHHH 135 132 9.9D VKKLL CFFFFLqFHP AAQPAMAHHHHHH 136 133 4.12F-f VKKLL CFSHLALPSP AAQPAMAHHHHHH 137 134 6.2B VKKLL FGSWIPFTQM AAQPAMAHHHHHH 138 135 4.6F VKKLL GLGYFNWTLL AAQPAMAHHHHHH 139 136 10.4A VKKLL HLFPLFQFHH AAQPAMAHHHHHH 140 137 5.6B VKKLL SEHVSSICVL AAQPAMAHHHHHH 141 138 3.11E VKKLL FSCLLDPTCP AAQPAMAHHHHHH 142 139 8.3F VKKLL LYLLHPSFLP AAQPAMAHHHHHH 143 140 2.2F VKKLL WCAPLLYSLR AAQPAMAHHHHHH 144 141 2.3F VKKLL FAMFPYTFqT AAQPAMAHHHHHH 145 142 10.5D VKKLL LPSLFYVESL AAQPAMAHHHHHH 146 143 8.8B VKKLL SLWLSSLSVL AAQPAMAHHHHHH 147 144 YJ2.17 VKKLL PHLWFLWSLK AAQPAMAHHHHHH 148 145 7.5B VKKLL ASDPVWYFLW AAQPAMAHHHHHH 149 146 10.12D VKKLL GLPLMGLqSL AAQPAMAHHHHHH 150 147 2.4H VKKLL PQLLLLRALS AAQPAMAHHHHHH 151 148 5.5D VKKLL APSAFSLHLF AAQPAMAHHHHHH 152 149 9.4C VKKLL FqLSSLFVPY AAQPAMAHHHHHH 153 150 4.5H VKKLL VPSFLSTMIE AAQPAMAHHHHHH 154 151 2.7B VKKLL ASPFFASYLW AAQPAMAHHHHHH 155 152 YJ2.23 VKKLL LQYLLSPIGY AAQPAMAHHHHHH 156 153 6.2D VKKLL VLSVPISAHH AAQPAMAHHHHHH 157 154 7.4A VKKLL MMqALSSLPE AAQPAMAHHHHHH 158 155 4.12B VKKLL MPAVLATRLT AAQPAMAHHHHHH 159 156 6.12E VKKLL PFTAWIIDGW AAQPAMAHHHHHH 160 157 YJ2.125 VKKLL TQLLPLWQPL AAQPAMAHHHHHH 161 158 YJ2.21 VKKLL LVPSLLPLTQ AAQPAMAHHHHHH 162 159 10.12B VKKLL PIqSCMVIPS AAQPAMAHHHHHH 163 160 YJ2.35 VKKLL WSLHLATRLL AAQPAMAHHHHHH 164 161 6.11H VKKLL qQVLLCSTLR AAQPAMAHHHHHH 165 162 7.3B VKKLL LLRYFLDPMY AAQPAMAHHHHHH 166 163 10.12A VKKLL IPQFLRSHHR AAQPAMAHHHHHH 167 164 YJ2.6 VKKLL GVLHLALSLR AAQPAMAHHHHHH 168 165 4.12C VKKLL LVTSqFSLVP AAQPAMAHHHHHH 169 166 YJ2.19 VKKLL PLALSWFQLR AAQPAMAHHHHHH 170 167 YJ2.88 VKKLL QHQWYPTVLM AAQPAMAHHHHHH 171 168 YJ2.29 VKKLL LMYWLSKPLS AAQPAMAHHHHHH 172 169 YJ2.8 VKKLL TQLTLSSSPI AAQPAMAHHHHHH 173 170 YJ2.94 VKKLL QLTALLSRLI AAQPAMAHHHHHH 174 171 YJ2.107 VKKLL LMTFGTTPQS AAQPAMAHHHHHH 175 172 YJ2.133 VKKLL SAFSFSLSST AAQPAMAHHHHHH 176 173 6.1A VKKLL APWLVLPHFP AAQPAMAHHHHHH 177 174 YJ2.81 VKKLL HVLSFAPPMP AAQPAMAHHHHHH 178 175 YJ2.38 VKKLL NWLFFAHPFS AAQPAMAHHHHHH 179 176 YJ2.20 VKKLL QLAVLLGSLR AAQPAMAHHHHHH 180 177 7.1D VKKLL LFGLFYFRAC AAQPAMAHHHHHH 181 178 YJ2.98 VKKLL FQFFVVWRLL AAQPAMAHHHHHH 182 179 YJ2.39 VKKLL PWAWPPPPFW AAQPAMAHHHHHH 183 180 YJ2.130 VKKLL LQLVIVYYLR AAQPAMAHHHHHH 184 181 YJ2.16 VKKLL RQSVLLSALH AAQPAMAHHHHHH 185 182 3.12E VKKLL VYGYFLTTFR AAQPAMAHHHHHH 186 183 YJ2.53 VKKLL CFSPLFGFHT AAQPAMAHHHHHH 187 184 YJ2.100 VKKLL PGYALWQTIP AAQPAMAHHHHHH 188 185 YJ2.58 VKKLL QRIFICFFLR AAQPAMAHHHHHH 189 186 8.2A VKKLL PHVFSCqLSA AAQPAMAHHHHHH 190 187 5.10A VKKLL SPLSLSVKLL AAQPAMAHHHHHH 191 188 9.2D VKKLL ARSLFSGSML AAQPAMAHHHHHH 192 189 YJ2.92 VKKLL LQFLIVFPLR AAQPAMAHHHHHH 193 190 YJ2.32 VKKLL LAVLLGQSLR AAQPAMAHHHHHH 194 191 YJ2.14 VKKLL LLSHLFLRLH AAQPAMAHHHHHH 195 192 8.4E VKKLL LAMVFFVTLR AAQPAMAHHHHHH 196 193 YJ2.117 VKKLL WLFALPQENV AAQPAMAHHHHHH 197 194 YJ2.66 VKKLL HPLVLLSSSP AAQPAMAHHHHHH 198 195 YJ2.131 VKKLL LQYLFMLSMR AAQPAMAHHHHHH 199 196 4.11H VKKLL PALLIRYASV AAQPAMAHHHHHH 200 197 YJ2.78 VKKLL QQFTSPFLLL AAQPAMAHHHHHH 201 198 YJ2.44 VKKLL SPCFFLLYLR AAQPAMAHHHHHH 202 199 YJ2.90 VKKLL PGMPLFFTNS AAQPAMAHHHHHH 203 200 YJ2.47 VKKLL PQVFFLFRPF AAQPAMAHHHHHH 204 201 YJ2.110 VKKLL PFPILLQSPF AAQPAMAHHHHHH 205 202 YJ2.74 VKKLL FQACCLFPLQ AAQPAMAHHHHHH 206 203 YJ2.55 VKKLL AVVHTMPLFS AAQPAMAHHHHHH 207 204 YJ2.108 VKKLL QFSWAFVSIL AAQPAMAHHHHHH 208 205 YJ2.96 VKKLL PVCLFWSFFR AAQPAMAHHHHHH 209 206 YJ2.70 VKKLL QLLWQQQVPV AAQPAMAHHHHHH 210 207 YJ2.60 VKKLL PLQALSWFLR AAQPAMAHHHHHH 211 208 YJ2.119 VKKLL FYLLCRLSLQ AAQPAMAHHHHHH 212 209 YJ2.82 VKKLL YLQILVICLR AAQPAMAHHHHHH 213 210 YJ2.63 VKKLL QLFLIVFPLR AAQPAMAHHHHHH 214 211 10.5A VKKLL PLHFALFFRL AAQPAMAHHHHHH 215 212 YJ2.85 VKKLL PFPMHLVLPF AAQPAMAHHHHHH 216 213 YJ2.86 VKKLL PLLFSPPSLH AAQPAMAHHHHHH 217 214 YJ2.126 VKKLL CQSITFSSIW AAQPAMAHHHHHH 218 215 YJ2.112 VKKLL WQRLFPFLLI AAQPAMAHHHHHH 219 216 YJ2.77 VKKLL MVPFWPFSFT AAQPAMAHHHHHH 220 217 YJ2.103 VKKLL QAFPLPPLLV AAQPAMAHHHHHH 221 218 YJ2.134 VKKLL PLYLLFRSFV AAQPAMAHHHHHH 222 219 YJ2.91 VKKLL HRSMYLSWLY AAQPAMAHHHHHH 223 220 YJ2.64 VKKLL LLSTLVRAPY AAQPAMAHHHHHH 224 221 YJ2.87 VKKLL PLALSQWFLR AAQPAMAHHHHHH 225 222 YJ2.116 VKKLL AQGMIFFLRL AAQPAMAHHHHHH 226 223 YJ2.62 VKKLL FCCRLALQFF AAQPAMAHHHHHH 227 224 YJ2.102 VKKLL YLQFLSLMLS AAQPAMAHHHHHH 228 225 YJ2.106 VKKLL CQATFPTLLC AAQPAMAHHHHHH 229 226 YJ2.124 VKKLL ARSYLYFSLS AAQPAMAHHHHHH 230 227 YJ2.111 VKKLL YQSSFLPLFW AAQPAMAHHHHHH 231 228 YJ2.104 VKKLL SASFLAFRIT AAQPAMAHHHHHH 232 229 YJ2.67 VKKLL SVLFLSHYHS AAQPAMAHHHHHH 233 230 YJ2.105 VKKLL PLALLYVRLS AAQPAMAHHHHHH 234 231 YJ2.127 VKKLL PEFLLLFRFF AAQPAMAHHHHHH 235 232 YJ2.80 VKKLL FPSLYAWGGL AAQPAMAHHHHHH 236 233 YJ2.122 VKKLL LQAAAFFCWL AAQPAMAHHHHHH 237 234 YJ2.79 VKKLL PFFLFCSSLR AAQPAMAHHHHHH 238 235 YJ2.115 VKKLL ELTQLWLFHL AAQPAMAHHHHHH 239 236 YJ2.113 VKKLL PGVPLLLCFR AAQPAMAHHHHHH 240 237 YJ2.114 VKKLL SQAYLSYFLY AAQPAMAHHHHHH 241 238 YJ2.61 VKKLL ISYAFLVRVT AAQPAMAHHHHHH 242 239 YJ2.123 VKKLL APALLRSILA AAQPAMAHHHHHH 243 240 YJ2.109 VKKLL HSHTLLMSLH AAQPAMAHHHHHH 244 241 YJ2.83 VKKLL AVSAFVSLVR AAQPAMAHHHHHH 245 242 YJ2.31 VKKLL TLITFKFLPH AAQPAMAHHHHHH 246 243 YJ2.49 VKKLL QQFAIPLVEF AAQPAMAHHHHHH 247 244 YJ2.75 VKKLL MPCLLVYYLE AAQPAMAHHHHHH 248 245 YJ2.71 VKKLL RYCLLLQIVR AAQPAMAHHHHHH 249 246 YJ2.45 VKKLL SLALLRVSLG AAQPAMAHHHHHH 250

247 YJ2.68 VKKLL IIGRIALILR AAQPAMAHHHHHH 251 248 YJ2.24 VKKLL PQLICAFILR AAQPAMAHHHHHH 252 249 8.3E VKKLL MVPLFPLPLP AAQPAMAHHHHHH 253 250 8.1B VKKLL HqAILYYYLN AAQPAMAHHHHHH 254

TABLE-US-00003 TABLE 3 Preference sequence patterns selected from L3 S5 sc-dsFv library SEQ ID No. Code Sequence NO M13-pe1B VKKLLFAIPL VVPFYAAQPA MAHHHHHH 4 1 2.1A VKKLLFAIPL LPAQAMPMSR MAHHHHHH * 255 2 7.5C VKKLLFAIPL YFVLVRESSS MAHHHHHH * 256 3 1.3B VKKLLFAIPL VLVVSSRTRA MAHHHHHH * 257 4 YJ3.25 VKKLLFAIPL LLSRPRAVPD MAHHHHHH * 258 5 3.8A VKKLLFAIPL CVSVRSPAFA MAHHHHHH * 259 6 1.6A VKKLLFAIPL MTTLASRTHA MAHHHHHH * 260 7 1.4H VKKLLFAIPL YLSMTRSGAA MAHHHHHH * 261 8 7.8F VKKLLFAIPL WLRSSVPVDS MAHHHHHH * 262 9 7.8H VKKLLFAIPL LSSLTRDSSS MAHHHHHH * 263 10 7.5E VKKLLFAIPL GLFTIRDSFA MAHHHHHH * 264 11 7.6C VKKLLFAIPL WLGITKPVWS MAHHHHHH * 265 12 1.3F VKKLLFAIPL YTLTPRPVFS MAHHHHHH * 266 13 1.5F VKKLLFAIPL qLALSRPSFP MAHHHHHH * 267 14 14.9A VKKLLFAIPL SSFLVADQSS MAHHHHHH * 268 15 YJ3.7 VKKLLFAIPL LLGLASPRSR MAHHHHHH * 269 16 13.1E VKKLLFAIPL LTLSNRSAWS MAHHHHHH * 270 17 2.2C VKKLLFAIPL LSLYPTRSTA MAHHHHHH * 271 18 YJ3.10 VKKLLFAIPL LTTLSRPSFS MAHHHHHH * 272 19 8.1A VKKLLFAIPL YFSRPPqPSS MAHHHHHH * 273 20 6.2H VKKLLFAIPL TMSSPPRSTS MAHHHHHH * 274 21 8.1C VKKLLFAIPL YFLRISPSAS MAHHHHHH * 275 22 1.8B VKKLLFAIPL LFLRPSAARP MAHHHHHH * 276 23 1.8C VKKLLFAIPL LWSSSRPTSQ MAHHHHHH * 277 24 YJ3.41 VKKLLFAIPL YLVCSRPLHA MAHHHHHH * 278 25 10.8G VKKLLFAIPL VLQRPPSPNT MAHHHHHH * 279 26 2.7C VKKLLFAIPL AMASFRPRDQ MAHHHHHH * 280 27 7.10C VKKLLFAIPL SRSLAMQPLP MAHHHHHH * 281 28 1.2A VKKLLFAIPL LSSLRSSNPE MAHHHHHH * 282 29 YJ3.4 VKKLLFAIPL SILINFRASS MAHHHHHH * 283 30 1.6B VKKLLFAIPL YWRSFWEPPA MAHHHHHH * 284 31 4.8E VKKLLFAIPL YLAAPRSTVA MAHHHHHH * 285 32 6.7H VKKLLFAIPL QYSAFSMSPR MAHHHHHH * 286 33 7.9C VKKLLFAIPL YLVSSKNSYP MAHHHHHH * 287 34 YJ3.72 VKKLLFAIPL GLSVSFRTSA MAHHHHHH * 288 35 4.4C VKKLLFAIPL AMLEPTRSSA MAHHHHHH * 289 36 11.1B VKKLLFAIPL SLSLHRPALA MAHHHHHH * 290 37 6.6B VKKLLFAIPL LSASARGSYA MAHHHHHH * 291 38 YJ3.26 VKKLLFAIPL YLAVTHRAYS MAHHHHHH * 292 39 YJ3.44 VKKLLFAIPL FFSLSRYSLA MAHHHHHH * 293 40 5.4B VKKLLFAIPL YLSAPRHASP MAHHHHHH * 294 41 5.2D VKKLLFAIPL WSFSRLPSSD MAHHHHHH * 295 42 12.4E VKKLLFAIPL YLSLTKPSLS MAHHHHHH * 296 43 14.1C VKKLLFAIPL SSPATEVLSP MAHHHHHH * 297 44 6.2C VKKLLFAIPL TLFLQRSSLA MAHHHHHH * 298 45 YJ3.6 VKKLLFAIPL VFTRVPHKPS MAHHHHHH * 299 46 4.1E VKKLLFAIPL AITRSSQFPS MAHHHHHH * 300 47 6.4H VKKLLFAIPL LGDLRSSPDA MAHHHHHH * 301 48 YJ3.53 VKKLLFAIPL VTTLSTRCYA MAHHHHHH * 302 49 7.7B VKKLLFAIPL FDASLEGPAM MAHHHHHH * 303 50 11.3C VKKLLFAIPL YFSSPSSRAP MAHHHHHH * 304 51 1.12A VKKLLFAIPL WFSFPFRSAA MAHHHHHH 305 52 12.1A VKKLLFAIPL YLSMSSPARS MAHHHHHH 306 53 1.12D VKKLLFAIPL SWSLCRPVCA MAHHHHHH 307 54 4.3G VKKLLFAIPL LYCWPRHSWS MAHHHHHH 308 55 YJ3.38 VKKLLFAIPL IFYTTRSSLS MAHHHHHH 309 56 YJ3.45 VKKLLFAIPL IYTLRSHSMT MAHHHHHH 400 57 2.9H VKKLLFAIPL PVPSLLGSAD MAHHHHHH 401 58 9.5A VKKLLFAIPL SLSLNSRSYP MAHHHHHH 402 59 2.7H VKKLLFAIPL FSPTSQEIRH MAHHHHHH 403 60 2.2G VKKLLFAIPL YFSCPLRVAS MAHHHHHH 404 61 YJ3.81 VKKLLFAIPL VLSLNRGVFA MAHHHHHH 405 62 7.4H VKKLLFAIPL SPqVLSSSPG MAHHHHHH 406 63 4.2C VKKLLFAIPL YVNAMSSPRP MAHHHHHH 407 64 13.6D VKKLLFAIPL YFTFVRSSWC MAHHHHHH 408 65 5.8D VKKLLFAIPL FDLSSDSVSP MAHHHHHH 409 66 YJ3.47 VKKLLFAIPL YILFWRNTHA MAHHHHHH 410 67 13.7A VKKLLFAIPL SCFLSRSAFS MAHHHHHH 411 68 YJ3.83 VKKLLFAIPL FFMITSKSRS MAHHHHHH 412 69 12.6C VKKLLFAIPL IVSSSRGSFA MAHHHHHH 413 70 4.10B VKKLLFAIPL AASRPLSPAA MAHHHHHH 414 71 YJ3.46 VKKLLFAIPL WLFSPLRSYS MAHHHHHH 415 72 YJ3.56 VKKLLFAIPL FLSYVRPLSA MAHHHHHH 416 73 13.5G VKKLLFAIPL FIFTPRSVHS MAHHHHHH 417 74 2.2E VKKLLFAIPL VSSIYKNSPP MAHHHHHH 418 75 5.5H VKKLLFAIPL MSDSTAPSFA MAHHHHHH 419 76 6.4B VKKLLFAIPL TLPqPRFPSP MAHHHHHH 420 77 7.10G VKKLLFAIPL SLLADSPRRP MAHHHHHH 421 78 5.3A VKKLLFAIPL FTDNSGEPSL MAHHHHHH 422 79 11.1E VKKLLFAIPL YCMPMSRTCA MAHHHHHH 423 80 11.1D VKKLLFAIPL MSRLSYHTPS MAHHHHHH 424 81 2.2F VKKLLFAIPL LSNSRVPPSS MAHHHHHH 425 82 15.7A VKKLLFAIPL FFASMRHTqA MAHHHHHH 426 83 YJ3.5 VKKLLFAIPL LLSTIKTSFS MAHHHHHH 427 84 3.3A VKKLLFAIPL FQQSSLSSVP MAHHHHHH 428 85 16.11A VKKLLFAIPL TLILSHRSSA MAHHHHHH 429 86 11.12A VKKLLFAIPL SFSRDPSFTS MAHHHHHH 430 87 9.1B VKKLLFAIPL ALSPTRHTLA MAHHHHHH 431 88 13.9A VKKLLFAIPL NILFTVRVYA MAHHHHHH 432 89 YJ3.15 VKKLLFAIPL LASLSARCHG MAHHHHHH 433 90 12.6B VKKLLFAIPL SVTLSLRASA MAHHHHHH 434 91 15.8H VKKLLFAIPL SHDPLLLSSP MAHHHHHH 435 92 YJ3.71 VKKLLFAIPL LWSLSSRGMT MAHHHHHH 436 93 YJ3.82 VKKLLFAIPL LISYCRPVSS MAHHHHHH 437 94 9.1D VKKLLFAIPL HSVELPASPA MAHHHHHH 438 95 9.6A VKKLLFAIPL LLSTSRSSSG MAHHHHHH 439 96 YJ3.34 VKKLLFAIPL WFSCSRFALS MAHHHHHH 440 97 YJ3.28 VKKLLFAIPL VCTLSSRAFS MAHHHHHH 441 98 11.1H VKKLLFAIPL YSPLARNPFS MAHHHHHH 442 99 16.9D VKKLLFAIPL FFAFSRQSSG MAHHHHHH 443 100 YJ3.70 VKKLLFAIPL TFSIFSRALA MAHHHHHH 444 101 YJ3.55 VKKLLFAIPL SLFFSARAIA MAHHHHHH 445 102 9.7A VKKLLFAIPL SQPSLCDPVP MAHHHHHH 446 103 10.11A VKKLLFAIPL LASYHRVAFA MAHHHHHH 447 104 10.1F VKKLLFAIPL WQLWQLPSRP MAHHHHHH 448 105 16.8A VKKLLFAIPL FTPMYRPTSP MAHHHHHH 449 106 YJ3.27 VKKLLFAIPL LLSLHRFSFA MAHHHHHH 450 107 9.5H VKKLLFAIPL SYSHPQNALA MAHHHHHH 451 108 10.12D VKKLLFAIPL YVLRSDASWG MAHHHHHH 452 109 4.2D VKKLLFAIPL FSGPPFDRTS MAHHHHHH 453 110 YJ3.66 VKKLLFAIPL FCALSRFTHA MAHHHHHH 454 111 YJ3.24 VKKLLFAIPL FSLSRPVPPL MAHHHHHH 455 112 10.7D VKKLLFAIPL SMDSFSRPFF MAHHHHHH 456 113 15.7C VKKLLFAIPL YTIIPSRASS MAHHHHHH 457 114 15.12C VKKLLFAIPL VPSANPPPLS MAHHHHHH 458 115 15.7E VKKLLFAIPL YLIKPPEGFS MAHHHHHH 459 116 YJ3.42 VKKLLFAIPL ISTLHFRAFG MAHHHHHH 460 117 YJ3.37 VKKLLFAIPL VRVMCGHSYA MAHHHHHH 461 118 YJ3.67 VKKLLFAIPL VLSLSRTFSG MAHHHHHH 462 119 YJ3.75 VKKLLFAIPL WCALSRQSMP MAHHHHHH 463 120 YJ3.86 VKKLLFAIPL YFWSLRVSWP MAHHHHHH 464

121 YJ3.33 VKKLLFAIPL YILSPRLPPP MAHHHHHH 465 122 YJ3.22 VKKLLFAIPL VVAAHRFSYA MAHHHHHH 466 123 YJ3.62 VKKLLFAIPL YVHLTSKAIP MAHHHHHH 467 124 YJ3.59 VKKLLFAIPL SLTLYRSGWS MAHHHHHH 468 125 YJ3.18 VKKLLFAIPL YYALSGRPVT MAHHHHHH 469 126 YJ3.79 VKKLLFAIPL MLSLMRQSAP MAHHHHHH 470

TABLE-US-00004 TABLE 4 Preference sequence patterns selected from L4 S5 sc-dsFv library SEQ ID No. Code Sequence NO M13-pe1B VKKLLFAIPLVVPFY AAQPAMAHHH HHH 4 1 1.11A VKKLLFAIPLVVPFY ARPLTRIQTP HHH * 471 2 9.3D VKKLLFAIPLVVPFY LTQLSRREPS HHH * 472 3 1.6B VKKLLFAIPLVVPFY ARSLATSPSR HHH * 473 4 14.5H VKKLLFAIPLVVPFY PARSYMLVRP HHH * 474 5 12.2A VKKLLFAIPLVVPFY SRSYMLLSRP HHH * 475 6 12.6H VKKLLFAIPLVVPFY TRSALAFFLP HHH * 476 7 YJ4.13 VKKLLFAIPLVVPFY SRGFTLPRLI HHH * 477 8 YJ4.1 VKKLLFAIPLVVPFY SSAFTRPIRP HHH * 478 9 12.2E VKKLLFAIPLVVPFY TRYSHAFMLI HHH * 479 10 6.10B VKKLLFAIPLVVPFY ARPMSMFRSD HHH * 480 11 8.4D VKKLLFAIPLVVPFY ASSMSqYRQN HHH * 481 12 5.9H VKKLLFAIPLVVPFY ARSYSRPPSI HHH * 482 13 10.8A VKKLLFAIPLVVPFY ASSMSRLRPH HHH * 483 14 YJ4.3 VKKLLFAIPLVVPFY CRSLSRPMLV HHH * 484 15 4.6C VKKLLFAIPLVVPFY SRSMSLHPTA HHH * 485 16 CM11 VKKLLFAIPLVVPFY TRSMTRLAPP HHH * 486 17 9.8H VKKLLFAIPLVVPFY TRAMSVSHKT HHH * 487 18 13.1F VKKLLFAIPLVVPFY LLAPKPSVKR HHH * 488 19 9.7A VKKLLFAIPLVVPFY SRPAPALSRL HHH * 489 20 15.9C VKKLLFAIPLVVPFY AKAMSARYQS HHH * 490 21 CM18 VKKLLFAIPLVVPFY FASQRSSPIR HHH * 491 22 CM24 VKKLLFAIPLVVPFY CLSFTSARFq HHH * 492 23 12.1A VKKLLFAIPLVVPFY PSASSRLSPK HHH * 493 24 2.10G VKKLLFAIPLVVPFY ARSYTRVPLA HHH * 494 25 CM2 VKKLLFAIPLVVPFY ARSLTFLPPR HHH * 495 26 9.4C VKKLLFAIPLVVPFY TTRVNAFMLV HHH * 496 27 11.11H VKKLLFAIPLVVPFY QAFRPVPVRN HHH * 497 28 11.8H VKKLLFAIPLVVPFY TSGMSRLRSW HHH * 498 29 1.12C VKKLLFAIPLVVPFY SRSPSQLSSR HHH * 499 30 16.12H VKKLLFAIPLVVPFY AFSLSRTSSK HHH * 500 31 3.11F VKKLLFAIPLVVPFY FHRVQQFSPA HHH * 501 32 9.2B VKKLLFAIPLVVPFY LDSMLTFRRS HHH * 502 33 CM40 VKKLLFAIPLVVPFY CRSLTSPLRM HHH * 503 34 15.5B VKKLLFAIPLVVPFY SRSASFLRPI HHH * 504 35 9.2F VKKLLFAIPLVVPFY MTFqSNSPRG HHH * 505 36 CM38 VKKLLFAIPLVVPFY CRPMTLRqPV HHH * 506 37 CM5 VKKLLFAIPLVVPFY VRPMSRVIMS HHH * 507 38 CM36 VKKLLFAIPLVVPFY SYGFSRPFSK HHH * 508 39 11.9G VKKLLFAIPLVVPFY TRSCFAFMLP HHH * 509 40 6.8B VKKLLFAIPLVVPFY AFSGAFRQSQ HHH * 510 41 16.6B VKKLLFAIPLVVPFY LRAGSFSAAP HHH * 511 42 CM22 VKKLLFAIPLVVPFY SHSMAPPSRR HHH * 512 43 CM31 VKKLLFAIPLVVPFY CRSGTFGNIG HHH * 513 44 11.5F VKKLLFAIPLVVPFY ARSMASTPLA HHH * 514 45 YJ4.2 VKKLLFAIPLVVPFY VYPLAPRLRD HHH * 515 46 6.10H VKKLLFAIPLVVPFY SLPWRRTPFQ HHH * 516 47 10.3D VKKLLFAIPLVVPFY MRTPPLSqRI HHH * 517 48 CM28 VKKLLFAIPLVVPFY ARSLSSYNAV HHH * 518 49 12.4D VKKLLFAIPLVVPFY VHALARKSQF HHH * 519 50 CM25 VKKLLFAIPLVVPFY SRSFSSPSIT HHH 520 51 13.5A VKKLLFAIPLVVPFY CRALSKPLPP HHH 521 52 12.6C VKKLLFAIPLVVPFY CRPSAPKMLL HHH 522 53 CM16 VKKLLFAIPLVVPFY SRSMSYFqPL HHH 523 54 4.2C VKKLLFAIPLVVPFY TRSLSRSIPH HHH 524 55 16.6C VKKLLFAIPLVVPFY SQLHqSPGNP HHH 525 56 10.10A VKKLLFAIPLVVPFY TRAIARPPYT HHH 526 57 10.11G VKKLLFAIPLVVPFY ARSLSTVRFP HHH 527 58 CM8 VKKLLFAIPLVVPFY TRAFSSPLSN HHH 528 59 9.6D VKKLLFAIPLVVPFY NRTPTIqRDS HHH 529 60 8.4B VKKLLFAIPLVVPFY ARAVSRTVPT HHH 530 61 8.5E VKKLLFAIPLVVPFY AqSMAVPIST HHH 531 62 13.2C VKKLLFAIPLVVPFY PqPSRGFMLI HHH 532 63 CM10 VKKLLFAIPLVVPFY TRSMVFPAKV HHH 533 64 CM26 VKKLLFAIPLVVPFY SRSMTLKGPE HHH 534 65 CM17 VKKLLFAIPLVVPFY AFPFSRQPNA HHH 535 66 CM7 VKKLLFAIPLVVPFY SRALTSISGM HHH 536 67 CM6 VKKLLFAIPLVVPFY CRGMSLNVTR HHH 537 68 6.10C VKKLLFAIPLVVPFY SHWRTQRPPE HHH 538 69 CM45 VKKLLFAIPLVVPFY ARSFSSPPGP HHH 539 70 13.1G VKKLLFAIPLVVPFY IFPIEASARR HHH 540 71 CM39 VKKLLFAIPLVVPFY ASSMALRPRV HHH 541 72 YJ4.74 VKKLLFAIPLVVPFY SRAFSSTPAM HHH 542 73 1.7F VKKLLFAIPLVVPFY SRSMVLQGPT HHH 543 74 YJ4.28 VKKLLFAIPLVVPFY SRSMTSPPYI HHH 544 75 10.3B VKKLLFAIPLVVPFY ANRPQSTKNI HHH 545 76 YJ4.56 VKKLLFAIPLVVPFY SRALTMTPSF HHH 546 77 4.6H VKKLLFAIPLVVPFY PTRLFAFMLT HHH 547 78 14.12A VKKLLFAIPLVVPFY SRAMSPIPRQ HHH 548 79 CM29 VKKLLFAIPLVVPFY ARSMGSMWQL HHH 549 80 YJ4.42 VKKLLFAIPLVVPFY SFSMTRSSPL HHH 550 81 CM42 VKKLLFAIPLVVPFY SFSFTRqPLP HHH 551 82 YJ4.33 VKKLLFAIPLVVPFY NRVPSPASQT HHH 552 83 YJ4.23 VKKLLFAIPLVVPFY SFSFSKPRFS HHH 553 84 CM27 VKKLLFAIPLVVPFY ARSLTQFSSV HHH 554 85 YJ4.39 VKKLLFAIPLVVPFY ARCFSSPVAL HHH 555 86 11.3B VKKLLFAIPLVVPFY GASSWWLFPS HHH 556 87 YJ4.84 VKKLLFAIPLVVPFY TPPQQQALLS HHH 557 88 14.1F VKKLLFAIPLVVPFY SRGFSMAFFP HHH 558 89 CM33 VKKLLFAIPLVVPFY SLAMSRPqAS HHH 559 90 13.12C VKKLLFAIPLVVPFY TYALTTFqSV HHH 560 91 YJ4.44 VKKLLFAIPLVVPFY QHAFTRPFRV HHH 561 92 CM30 VKKLLFAIPLVVPFY SRAFSSPSGS HHH 562 93 13.11G VKKLLFAIPLVVPFY TSALARSPRV HHH 563 94 4.8B VKKLLFAIPLVVPFY CRAMSSPFRP HHH 564 95 4.2B VKKLLFAIPLVVPFY STFARSFMLT HHH 565 96 9.2D VKKLLFAIPLVVPFY FPLSSRAFML HHH * 566 97 YJ4.71 VKKLLFAIPLVVPFY SRSMSTSPIL HHH 567 98 9.6H VKKLLFAIPLVVPFY SFGLqLPqPF HHH 568 99 CM37 VKKLLFAIPLVVPFY SRSMSLSSDL HHH 569 100 16.3E VKKLLFAIPLVVPFY AFPLARRPIN HHH 570 101 12.1B VKKLLFAIPLVVPFY TSCRAMTLPR HHH 571 102 CM23 VKKLLFAIPLVVPFY TYPFSRAGPP HHH 572 103 YJ4.47 VKKLLFAIPLVVPFY ANQQALPFQL HHH 573 104 YJ4.38 VKKLLFAIPLVVPFY GWSMSLRSHS HHH 574 105 4.11H VKKLLFAIPLVVPFY SPQVVTRKDL HHH 575 106 12.9G VKKLLFAIPLVVPFY LRNAHAMASA HHH 576 107 CM44 VKKLLFAIPLVVPFY SRSGSFNVTP HHH 577 108 11.3E VKKLLFAIPLVVPFY SRPLSRVPVF HHH 578 109 11.9F VKKLLFAIPLVVPFY SKRMPPPISq HHH 579 110 CM34 VKKLLFAIPLVVPFY TRSMSSLPSP HHH 580 111 14.11D VKKLLFAIPLVVPFY CRSSSSIFPL HHH 581 112 CM15 VKKLLFAIPLVVPFY RSAHAMSIQT HHH 582 113 10.1H VKKLLFAIPLVVPFY GYCFSARIIR HHH 583 114 9.10A VKKLLFAIPLVVPFY PHLSPLqPQq HHH 584 115 CM43 VKKLLFAIPLVVPFY SFSFSRFPGL HHH 585 116 YJ4.48 VKKLLFAIPLVVPFY SSSMSLRPQF HHH 586 117 11.11D VKKLLFAIPLVVPFY SSPRARPVPP HHH 587 118 CM46 VKKLLFAIPLVVPFY ARSLSALSPY HHH 588 119 12.5C VKKLLFAIPLVVPFY PVRqLHTNLR HHH 589 120 10.2F VKKLLFAIPLVVPFY PTTSTPYqSP HHH 590

121 CM21 VKKLLFAIPLVVPFY VNALTFLPSq HHH 591 122 CM41 VKKLLFAIPLVVPFY ARSLSSPLTL HHH 592 123 YJ4.25 VKKLLFAIPLVVPFY TRPPTVGLRQ HHH 593 124 CM14 VKKLLFAIPLVVPFY TRALSPMSWq HHH 594 125 YJ4.6 VKKLLFAIPLVVPFY VFPFSRPLLR HHH 595 126 CM1 VKKLLFAIPLVVPFY VPRCLSMSLG HHH 596 127 YJ4.87 VKKLLFAIPLVVPFY QQPSFHPISR HHH 597 128 CM32 VKKLLFAIPLVVPFY SKAFSSFqAS HHH 598 129 10.6H VKKLLFAIPLVVPFY GYSMSqSGLT HHH 599 130 YJ4.40 VKKLLFAIPLVVPFY AQALTTRGLA HHH 600 131 YJ4.26 VKKLLFAIPLVVPFY VKSLTRPAFL HHH 601 132 12.4F VKKLLFAIPLVVPFY AqSRLRVYPP HHH 602 133 4.5B VKKLLFAIPLVVPFY PAIGFMLLRY HHH 603 134 12.3D VKKLLFAIPLVVPFY SFGTLVRPRP HHH 604 135 CM3 VKKLLFAIPLVVPFY IRRPVDPVMP HHH 605 136 YJ4.19 VKKLLFAIPLVVPFY FPLRQTHRYP HHH 606 137 13.2H VKKLLFAIPLVVPFY THSMQRPTGR HHH 607 138 10.5D VKKLLFAIPLVVPFY RHTqLSSSTS HHH 608 139 15.10D VKKLLFAIPLVVPFY SCGFSRLSKA HHH 609 140 CM35 VKKLLFAIPLVVPFY SRSFSQLPHI HHH 610 141 YJ4.43 VKKLLFAIPLVVPFY SSSMSQLRPF HHH 611 142 10.2B VKKLLFAIPLVVPFY CRTTFALQSS HHH 612 143 CM19 VKKLLFAIPLVVPFY AQSMSIRHNN HHH 613 144 11.4E VKKLLFAIPLVVPFY NSRFRTTPPS HHH 614 145 CM20 VKKLLFAIPLVVPFY SVSMSRYQLS HHH 615 146 CM12 VKKLLFAIPLVVPFY SSGASRLRIL HHH 616 147 YJ4.81 VKKLLFAIPLVVPFY CWSLSRPRLL HHH 617 148 10.1C VKKLLFAIPLVVPFY TSRSTKLTPS HHH 618 149 11.6D VKKLLFAIPLVVPFY SRVSVAFMLM HHH 619 150 YJ4.72 VKKLLFAIPLVVPFY CLGRSMAPGP HHH 620 151 14.1A VKKLLFAIPLVVPFY FVHRRDSSSL HHH 621 152 YJ4.24 VKKLLFAIPLVVPFY SLGFSRLTSL HHH 622 153 13.2B VKKLLFAIPLVVPFY ASALSRRVPq HHH 623 154 11.6B VKKLLFAIPLVVPFY TYPASWPRLR HHH 624 155 9.2G VKKLLFAIPLVVPFY SRVSLAVTPS HHH 625 156 10.11B VKKLLFAIPLVVPFY NNPFSSLSqq HHH 626 157 11.8D VKKLLFAIPLVVPFY RPLPRPFAGN HHH 627 158 CM4 VKKLLFAIPLVVPFY GFSMTQYLPq HHH 628 159 YJ4.75 VKKLLFAIPLVVPFY SSALSRSFYP HHH 629 160 YJ4.61 VKKLLFAIPLVVPFY TQQRCFAMHI HHH 630 161 YJ4.85 VKKLLFAIPLVVPFY IKHFYNSRPS HHH 631 162 YJ4.51 VKKLLFAIPLVVPFY FTRLPKESSP HHH 632 163 9.6G VKKLLFAIPLVVPFY LPAQPRVTRT HHH 633 164 CM13 VKKLLFAIPLVVPFY LRSMTLNTST HHH 634 165 YJ4.35 VKKLLFAIPLVVPFY PDTFSYSSQD HHH 635 166 YJ4.41 VKKLLFAIPLVVPFY FRNPQLPSSA HHH 636 167 YJ4.50 VKKLLFAIPLVVPFY FRPDRTPPSS HHH 637 168 9.8C VKKLLFAIPLVVPFY qSHTILPLPA HHH 638 169 CM9 VKKLLFAIPLVVPFY SSAFqPMVSS HHH 639 170 9.7H VKKLLFAIPLVVPFY QSRRLPILPL HHH 640 171 YJ4.31 VKKLLFAIPLVVPFY GQAYLPAPQL HHH 641 172 9.11B VKKLLFAIPLVVPFY TSRPRETLFL HHH 642 173 9.3G VKKLLFAIPLVVPFY TAASVVRSRD HHH 643 174 10.5F VKKLLFAIPLVVPFY VRGAAPKFSV HHH 644 175 YJ4.14 VKKLLFAIPLVVPFY FRHQPASVST HHH 645 176 9.8B VKKLLFAIPLVVPFY PTNAIAFFLq HHH 646 177 YJ4.59 VKKLLFAIPLVVPFY LKSLRSDTPN HHH 647 178 YJ4.22 VKKLLFAIPLVVPFY IKRPLPLAPT HHH 648 179 11.11F VKKLLFAIPLVVPFY ASSSKSRFML HHH 649 180 YJ4.82 VKKLLFAIPLVVPFY PWKPRLLPPQ HHH 650 181 9.1H VKKLLFAIPLVVPFY SRGFMLTLRY HHH 651 182 9.8E VKKLLFAIPLVVPFY CKARGIMPVF HHH 652 183 YJ4.17 VKKLLFAIPLVVPFY ASLPRLTSQS HHH 653 184 11.2B VKKLLFAIPLVVPFY qSSAFSYMLS HHH 654 185 10.7A VKKLLFAIPLVVPFY SFSSQRFLRP HHH 655 186 9.7G VKKLLFAIPLVVPFY TSSNTSRRFP HHH 656 187 11.10B VKKLLFAIPLVVPFY NqTAATAPPR HHH 657 188 10.8G VKKLLFAIPLVVPFY GAPLSWRRSY HHH 658 189 9.10D VKKLLFAIPLVVPFY CRSVWCIPRP HHH 659 190 9.1C VKKLLFAIPLVVPFY AKACLRPLQT HHH 660 191 9.6F VKKLLFAIPLVVPFY CLASSHRHRP HHH 661 192 11.3H VKKLLFAIPLVVPFY LRADSLAPKS HHH 662 193 9.9F VKKLLFAIPLVVPFY SVPQFSGRSR HHH 663 194 YJ4.78 VKKLLFAIPLVVPFY VYPARFPAKT HHH 664 195 YJ4.21 VKKLLFAIPLVVPFY NFMLRHPQTF HHH 665 196 YJ4.32 VKKLLFAIPLVVPFY YVPRFPPKSA HHH 666 197 YJ4.86 VKKLLFAIPLVVPFY LSPMSRTRYV HHH 667 198 YJ4.66 VKKLLFAIPLVVPFY TYPLTKPYRP HHH 668 199 YJ4.83 VKKLLFAIPLVVPFY SSYWSHRKPP HHH 669 200 10.8C VKKLLFAIPLVVPFY SPRTFAFFLM HHH 670 201 11.1A VKKLLFAIPLVVPFY LGPGIRKKPA HHH 671 202 9.4E VKKLLFAIPLVVPFY TRLCVAKVAG HHH 672 203 11.2E VKKLLFAIPLVVPFY RSLPASGASR HHH 673 204 10.5E VKKLLFAIPLVVPFY ASPRVKSYSP HHH 674 205 9.10F VKKLLFAIPLVVPFY PSRTFAFYLV HHH 675 206 9.4H VKKLLFAIPLVVPFY qqEFAMAHHH HHH 676 207 11.8B VKKLLFAIPLVVPFY PqSSKAFFLN HHH 677 208 11.2F VKKLLFAIPLVVPFY VKALRGSYPT HHH 678 209 11.7F VKKLLFAIPLVVPFY TqPSqVRYML HHH 679 210 11.9C VKKLLFAIPLVVPFY SARGqHVRPP HHH 680 211 10.11C VKKLLFAIPLVVPFY STRCPGFFLq HHH 681 212 11.6E VKKLLFAIPLVVPFY CPSVFSRTPP HHH 682 213 11.3A VKKLLFAIPLVVPFY DASSWRHFLS HHH 683

Example 4

Production of sc-dsFv Against H5 of Influenza Virus and Microarray Test

[0089] As described above, scFvs (8a and 12a) and their disulfide forms (ds-8a and ds-12a, respectively) to various hemagglutins (HAs) from different serotypes of influenza virus were developed. As shown in FIG. 5, the results indicated that selected scFv phage clones against H5 of influenza virus could be introduced to sc-dsFv directly but had lower binding affinity as compared with original scFvs. These results also suggested that the binding affinity could be enhanced by sc-dsFv phage panning procedures with the signal sequences described above.

[0090] The 8aS5 protein could be concentrated to 6 mg/ml without precipitation. The array studies suggested that 4 ng/spot of ds-8a protein could detect .about.10.sup.7 viruses in solution by using 40 nm fluorescence beads. In conclusion, the signal sequence derived from sc-dsFv phage production against VEGF from monoclonal antibody could be applied for sc-dsFv phage production against hemagglutinin from natural antibody repertoire. The binding affinity could be enhanced by sc-dsFv phage panning procedures to produce sc-dsFv with high binding capacity and better stability than scFv for further applications.

Example 5

Soluble Non-fusion sc-dsFv Expressed with Suppressor E. coli Strain

[0091] The signal sequences resulting in the successful expression of the displayed sc-dsFv on phage rescued from suppressor E. coli strain ER2738 were more likely to result in secretion of the soluble non-fusion anti-VEGF sc-dsFv in a culture medium. Signal sequence phage library L4 was selected for binding to immobilized VEGF and the VEGF-binding enriched phage variants were amplified for the next round of selection/amplification cycle. The selection/amplification cycle was repeated for four rounds. After each round of selection/amplification cycle, a random collection of 96 phage variants were picked from the amplified phage population. These phage variants were used to infect E. coli ER2738 and the soluble sc-dsFv was expressed in the overnight cultures, which were tested for binding to immobilized VEGF with ELISA.

[0092] These random collections of phage variants were also used to infect E. coli HB2151 for the same assay to determine the sc-dsFv secretion. The result showed that, with ER2738 as the host, 0%, 0%, 2%, and 14% of the phage variants from 1.sup.st, 2.sup.nd, 3.sup.rd, and 4.sup.th round of selection/amplification cycle respectively secreted functional sc-dsFv binding to VEGF with ELISA signal greater than OD.sub.450nm>0.6. But this trend was not found in the experiment with E. coli strain HB2151. This result indicated that signal sequence alteration could restore the secretion of the soluble non-fusion sc-dsFv and that the search for the optimum signal sequences could be facilitated with phage-based selection/amplification cycles on signal sequence libraries. This conclusion is applicable only to the E. coli suppressor strain ER2738 as the bacteria host for the M13 phage.

Example 6

Interface Disulfide Bond Formation in the sc-dsFv

[0093] One measurement for the folding quality of the sc-dsFv is the extent of the interface disulfide bond formation in the sc-dsFv. This measurement was determined by the ratio of the sc-dsFv-VEGF binding ELISA signal after the fXa (bovine factor Xa) treatment over that before the fXa treatment. FXa cleaves substrate sequence -IEGR- in the linker peptide connecting the two variable domains in the sc-dsFv construct. If the interface disulfide bond was not formed in the sc-dsFv, the cleavage of the linker peptide would result in dissociation of the variable domains and abolishment of the affinity against VEGF. Hence the ratio reflects the percentage of interface disulfide bond formation in the sc-dsFv. This measurement was validated with the positive control (anti-VEGF scFv(fXa+)/M13pIII-pelB with -IEGR- in the linker peptide but without the interface disulfide bond) and the negative control (anti-VEGF scFv(fXa-)/M13pIII-pelB without both the fXa cutting site and the interface disulfide bond).

[0094] FIG. 6A compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the disulfide bond formation in the sc-dsFv displayed on phage surface for the signal sequence variants from the L4 library. Strong correlation between the two measurement is evident (R.sup.2=0.508, p-value=0.000158). As shown in FIG. 6A, signal sequence optimization could improve the disulfide bond formation in the sc-dsFv from .about.0% up to 40% of the secreted sc-dsFv molecule.

[0095] Another folding quality of the sc-dsFv was determined by the ratio of the normalized sc-dsFv-VEGF binding ELISA signal over the normalized quantity of the secreted sc-dsFv determined by electrophoresis and Western blot analysis. FIG. 6B compared the extent of the interface disulfide bond formation in the secreted soluble sc-dsFv with the folding qualities derived from electrophoresis and ELISA measurements for the signal sequence variants from the L4 library. The positive correlation (R.sup.2=0.296, p-value=0.062) shown in FIG. 6B indicated that the interface disulfide bond formation enhanced the affinity for the sc-dsFv-VEGF interaction. The plot also indicated that the selected variants resulted in secreted sc-dsFv with up to more than 10-fold VEGF-binding signals per unit quantity of secreted sc-dsFv compared with the positive control scFv(fXa+)/M13pIII-pelB, indicating that the secreted sc-dsFv from these signal sequence variants folded into antibody-like structure substantially more effectively that the scFv construct. This is most likely due to the stabilizing interface disulfide bond that is formed in the sc-dsFv but is absent in the scFv construct.

Example 7

Correlation Between the Stability of sc-dsFv and the Extent of the Interface Disulfide Bond Formation in the sc-dsFv

[0096] The effect of interface disulfide bond in stabilizing the sc-dsFv structure was demonstrated in FIG. 7. Secreted sc-dsFv from representative variants selected from each of the three libraries were expressed and incubated at 37.degree. C. for 12 days and the affinities of the sc-dsFv's against VEGF were measured along the course of incubation. FIG. 7A shows the VEGF-binding affinity plotted against the time course of incubation for each of the selected variants. The VEGF affinity for the control anti-VEGF scFv dropped rapidly in the first few days of incubation, while a few variants from L4 library resulted in stable secreted sc-dsFv that were even gaining affinities against VEGF compared with freshly prepared secreted protein, presumably due to the increasingly stabilized sc-dsFv with the formation of the interface disulfide bond. The correlation between the two measurements shown in FIG. 7B is strong (R.sup.2=0.867 p-value=0.023), indicating that the interface disulfide bond could be one of the most important factors in stabilizing the secreted sc-dsFv in the culture medium.

[0097] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

Sequence CWU 1

1

598130PRTartificialSynthetic polypeptide 1Val Lys Lys Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His Gly His 20 25 30230PRTartificialSynthetic polypeptide 2Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Met Ala His His His His His His Gly His 20 25 30330PRTartificialSynthetic polypeptide 3Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His His His Gly His 20 25 30428PRTartificialSynthetic polypeptide 4Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 25528PRTartificialSynthetic polypeptide 5Val Lys Lys Leu Leu Val Leu Ser His Leu Pro Phe Met Thr Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 25628PRTartificialSynthetic polypeptide 6Val Lys Lys Leu Leu Ser His Trp Leu Leu Ser Ser Gln Leu Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 25728PRTartificialSynthetic polypeptide 7Val Lys Lys Leu Leu Ala Met Ser Leu Ala Pro Ser Val Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 25828PRTartificialSynthetic polypeptide 8Val Lys Lys Leu Leu Trp Ser Leu Phe Phe Gln Gln Leu Asn Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 25928PRTartificialSynthetic polypeptide 9Val Lys Lys Leu Leu Leu Leu Ser Leu Leu Gln Arg Pro Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251028PRTartificialSynthetic polypeptide 10Val Lys Lys Leu Leu Leu Ser Ser Trp Leu Met Thr Arg Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251128PRTartificialSynthetic polypeptide 11Val Lys Lys Leu Leu Val Leu Ser His Phe Pro Ala Phe Val Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251228PRTartificialSynthetic polypeptide 12Val Lys Lys Leu Leu Pro Leu Leu Ser Leu Pro Leu Pro Pro Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251328PRTartificialSynthetic polypeptide 13Val Lys Lys Leu Leu Val Leu Thr Pro Met His Phe Ser Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251428PRTartificialSynthetic polypeptide 14Val Lys Lys Leu Leu Ile Leu Ala Leu Pro Gln Ser Tyr Pro Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251528PRTartificialSynthetic polypeptide 15Val Lys Lys Leu Leu Gln Ala Leu Tyr Phe Ser Leu Pro Ser Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251628PRTartificialSynthetic polypeptide 16Val Lys Lys Leu Leu Val Ser Ala Met Thr Ser Ala Ser Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251728PRTartificialSynthetic polypeptide 17Val Lys Lys Leu Leu Leu Pro Ala Ser Trp Leu Phe Gly Gln Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251828PRTartificialSynthetic polypeptide 18Val Lys Lys Leu Leu Trp Ser Leu Phe Phe Gln Gln Leu Asn Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 251928PRTartificialSynthetic polypeptide 19Val Lys Lys Leu Leu Phe Val Met Ala Leu Arg Ser Ser Ala Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252028PRTartificialSynthetic polypeptide 20Val Lys Lys Leu Leu Phe Leu Trp Pro Phe Tyr Asn Gly His Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252128PRTartificialSynthetic polypeptide 21Val Lys Lys Leu Leu Gln Ser Phe Tyr Leu Ser Leu Gln Leu Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252228PRTartificialSynthetic polypeptide 22Val Lys Lys Leu Leu Ser Leu Thr Phe Pro Phe Thr Ile His Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252328PRTartificialSynthetic polypeptide 23Val Lys Lys Leu Leu Trp Pro Val Leu Ser Pro Ser Leu Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252428PRTartificialSynthetic polypeptide 24Val Lys Lys Leu Leu Pro Trp Leu Phe Ser Thr Phe Pro Ser Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252528PRTartificialSynthetic polypeptide 25Val Lys Lys Leu Leu Ile Met Ser Ser Leu Pro Thr Leu Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252628PRTartificialSynthetic polypeptide 26Val Lys Lys Leu Leu Ile Met Ser Arg Val Leu Ala Pro Asp Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252728PRTartificialSynthetic polypeptide 27Val Lys Lys Leu Leu Phe Asp Phe Trp Phe Ser Ser Phe Leu Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252828PRTartificialSynthetic polypeptide 28Val Lys Lys Leu Leu Tyr Gly Gln Leu Met Leu Leu Ser Ser Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 252928PRTartificialSynthetic polypeptide 29Val Lys Lys Leu Leu Pro Trp Leu Phe Pro Phe His Ala Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253028PRTartificialSynthetic polypeptide 30Val Lys Lys Leu Leu Leu Val Met Thr Leu Ser Arg Gln Pro Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253128PRTartificialSynthetic polypeptide 31Val Lys Lys Leu Leu Ala Ser Ala Tyr Leu Tyr His Gly Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253228PRTartificialSynthetic polypeptide 32Val Lys Lys Leu Leu Pro Phe Phe Ala Gly Val Leu Gln His Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253328PRTartificialSynthetic polypeptide 33Val Lys Lys Leu Leu Ala Leu Ser Ser Pro Phe Phe His Ile Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253428PRTartificialSynthetic polypeptide 34Val Lys Lys Leu Leu Pro Thr Arg Gln Pro Met Met Tyr Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253528PRTartificialSynthetic polypeptide 35Val Lys Lys Leu Leu Gln Leu Leu Met Pro Phe Leu Asn Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253628PRTartificialSynthetic polypeptide 36Val Lys Lys Leu Leu Cys Ser Leu Gly Tyr Ala Cys Ile Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253728PRTartificialSynthetic polypeptide 37Val Lys Lys Leu Leu Leu Met Pro Trp Leu Phe Asn Ser Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253828PRTartificialSynthetic polypeptide 38Val Lys Lys Leu Leu Leu Asp Gln Leu Ala Tyr Ala Ala Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 253928PRTartificialSynthetic polypeptide 39Val Lys Lys Leu Leu Gln Ser Thr Val Phe Phe Ser Trp Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254028PRTartificialSynthetic polypeptide 40Val Lys Lys Leu Leu Leu Pro Trp Ala Leu Ser His Gln Val Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254128PRTartificialSynthetic polypeptide 41Val Lys Lys Leu Leu Ala Leu Thr Tyr Pro Ala Phe Leu Tyr Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254228PRTartificialSynthetic polypeptide 42Val Lys Lys Leu Leu Ala Met Ala Pro Pro Met Met Ser Met Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254328PRTartificialSynthetic polypeptide 43Val Lys Lys Leu Leu Trp Trp Ser Ser Leu Phe Ala Pro Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254428PRTartificialSynthetic polypeptide 44Val Lys Lys Leu Leu Gly Ser Phe Ile Leu Ala Arg Ser Met Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254528PRTartificialSynthetic polypeptide 45Val Lys Lys Leu Leu Met Val Leu Thr Ser Trp His Pro Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254628PRTartificialSynthetic polypeptide 46Val Lys Lys Leu Leu Phe Ser Leu Arg Phe Phe Phe Pro Ser Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254728PRTartificialSynthetic polypeptide 47Val Lys Lys Leu Leu Trp Leu Trp Ser Thr Pro Leu Phe Pro His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254828PRTartificialSynthetic polypeptide 48Val Lys Lys Leu Leu Pro Leu Leu Phe Ser Leu Asp Gly Asp Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 254928PRTartificialSynthetic polypeptide 49Val Lys Lys Leu Leu Ser Val Ser Leu Ser Ser Tyr Ser Phe Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255028PRTartificialSynthetic polypeptide 50Val Lys Lys Leu Leu Leu Asn Gly Thr Phe Ser Ala Gln Leu Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255128PRTartificialSynthetic polypeptide 51Val Lys Lys Leu Leu Trp His Val Leu Pro Tyr Leu Pro Asn Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255228PRTartificialSynthetic polypeptide 52Val Lys Lys Leu Leu Ser Ile Val Pro Leu Phe Ser Pro Gln Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255328PRTartificialSynthetic polypeptide 53Val Lys Lys Leu Leu Val Met Thr Ser Pro Met Leu Ala Pro Gly Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255428PRTartificialSynthetic polypeptide 54Val Lys Lys Leu Leu Val Leu Ser Leu Pro Ser Ile Ala Pro His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255528PRTartificialSynthetic polypeptide 55Val Lys Lys Leu Leu Gln Ser Leu Leu Leu Leu Arg Ala Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255628PRTartificialSynthetic polypeptide 56Val Lys Lys Leu Leu Phe Ser Leu Pro Val Phe Phe Asp Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255728PRTartificialSynthetic polypeptide 57Val Lys Lys Leu Leu Leu Leu Phe Ser Met Ala Arg Pro Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255828PRTartificialSynthetic polypeptide 58Val Lys Lys Leu Leu Thr Gln Ala Val Phe Pro Phe Thr Phe Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 255928PRTartificialSynthetic polypeptide 59Val Lys Lys Leu Leu Leu Ala Ser Trp Leu Phe Arg Ala Asp Met Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256028PRTartificialSynthetic polypeptide 60Val Lys Lys Leu Leu Pro Phe Leu Phe Pro Phe Pro Ser Pro Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256128PRTartificialSynthetic polypeptide 61Val Lys Lys Leu Leu Ala Leu Ser Ala Trp Ser Leu Ser Gln Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256228PRTartificialSynthetic polypeptide 62Val Lys Lys Leu Leu Ala Leu Leu Pro Leu Phe Pro Thr Gln His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256328PRTartificialSynthetic polypeptide 63Val Lys Lys Leu Leu Ala Ala Leu Ala Ser Phe Pro Pro Ala Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256428PRTartificialSynthetic polypeptide 64Val Lys Lys Leu Leu Leu Leu Met Pro Phe Leu Asn Gln Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256528PRTartificialSynthetic polypeptide 65Val Lys Lys Leu Leu Phe Thr Ser Gly Leu Lys Leu Val Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256628PRTartificialSynthetic polypeptide 66Val Lys Lys Leu Leu Leu Gln Pro Leu Leu Ser Ile Tyr Leu Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256728PRTartificialSynthetic polypeptide 67Val Lys Lys Leu Leu Leu Ser Ser Leu Trp Ser Ala Tyr Met Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256828PRTartificialSynthetic polypeptide 68Val Lys Lys Leu Leu Leu Leu Gly Gln Ser Leu Met His Phe Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 256928PRTartificialSynthetic polypeptide 69Val Lys Lys Leu Leu Pro Gln Leu Ala Met Ser Leu Pro Ser Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257028PRTartificialSynthetic polypeptide 70Val Lys Lys Leu Leu Tyr Glu Thr Met Leu Ser Ser Tyr Leu Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257128PRTartificialSynthetic polypeptide 71Val Lys Lys Leu Leu Ser Leu Tyr Tyr Phe Pro Leu Val Pro Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257228PRTartificialSynthetic polypeptide 72Val Lys Lys Leu Leu Gln Arg Thr Val Ala Ala Ala Tyr Phe Trp Ala1 5 10

15Ala Gln Pro Ala Met Ala His His His His His His 20 257328PRTartificialSynthetic polypeptide 73Val Lys Lys Leu Leu Phe Leu Thr Trp Leu Arg Tyr Gly Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257428PRTartificialSynthetic polypeptide 74Val Lys Lys Leu Leu Leu Leu Leu Thr Leu Met Gln Pro Thr Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257528PRTartificialSynthetic polypeptide 75Val Lys Lys Leu Leu Phe Asp Phe Phe Thr His Val His Leu Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257628PRTartificialSynthetic polypeptide 76Val Lys Lys Leu Leu Ala Leu Tyr Pro His Phe Val Ser Phe Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257728PRTartificialSynthetic polypeptide 77Val Lys Lys Leu Leu Leu Pro Tyr Ala Ile Gln Leu Phe Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257828PRTartificialSynthetic polypeptide 78Val Lys Lys Leu Leu Trp Phe Pro Leu His Ser Ser Leu Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 257928PRTartificialSynthetic polypeptide 79Val Lys Lys Leu Leu Pro Ala Leu Leu Leu Ala Thr Ala Ala Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258028PRTartificialSynthetic polypeptide 80Val Lys Lys Leu Leu Leu Ala Ser Val Ala Trp Asn Leu Asp Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258128PRTartificialSynthetic polypeptide 81Val Lys Lys Leu Leu Val Gly Ser Leu Leu Phe Trp Pro Gln Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258228PRTartificialSynthetic polypeptide 82Val Lys Lys Leu Leu Ser Pro Leu Leu Phe Leu Gln Asn Tyr Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258328PRTartificialSynthetic polypeptide 83Val Lys Lys Leu Leu Ser Tyr Trp Leu Asp Phe Ile Gln Val Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258428PRTartificialSynthetic polypeptide 84Val Lys Lys Leu Leu Val Pro Ser Phe Leu Leu Ser Pro Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258528PRTartificialSynthetic polypeptide 85Val Lys Lys Leu Leu Ser Leu Tyr Trp Leu Thr Ser Gln Pro Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258628PRTartificialSynthetic polypeptide 86Val Lys Lys Leu Leu Phe Ala Leu Ser Ser Val His Ser Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258728PRTartificialSynthetic polypeptide 87Val Lys Lys Leu Leu Ser Tyr Tyr Ser Leu Leu Tyr Ser Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258828PRTartificialSynthetic polypeptide 88Val Lys Lys Leu Leu Leu Val Ser Gly Leu Gln Pro Trp Tyr Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 258928PRTartificialSynthetic polypeptide 89Val Lys Lys Leu Leu Val Leu Ala Thr Pro Leu His Leu Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259028PRTartificialSynthetic polypeptide 90Val Lys Lys Leu Leu Ser Leu Ala Phe Pro Leu Phe Thr Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259128PRTartificialSynthetic polypeptide 91Val Lys Lys Leu Leu Ser Leu Val Pro Ile Phe Pro Phe Ser Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259228PRTartificialSynthetic polypeptide 92Val Lys Lys Leu Leu Gln Pro Val Leu Phe Ser Phe Phe Ile Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259328PRTartificialSynthetic polypeptide 93Val Lys Lys Leu Leu Met Ser Gln Phe Leu Asn Leu Leu Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259428PRTartificialSynthetic polypeptide 94Val Lys Lys Leu Leu Trp Ala Val Gln Pro Leu Phe Pro Leu Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259528PRTartificialSynthetic polypeptide 95Val Lys Lys Leu Leu Met Phe Ser Leu Val Pro Ser Pro Pro Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259628PRTartificialSynthetic polypeptide 96Val Lys Lys Leu Leu Pro Phe Phe Leu Gln Pro Phe Gln Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259728PRTartificialSynthetic polypeptide 97Val Lys Lys Leu Leu Pro Asp Leu Leu Ala Ser Val Leu Pro Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259828PRTartificialSynthetic polypeptide 98Val Lys Lys Leu Leu Phe Trp Gln Phe Leu Trp Pro Ser Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 259928PRTartificialSynthetic polypeptide 99Val Lys Lys Leu Leu Leu Leu Gly Gln Phe Phe Pro Asn Pro Met Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510028PRTartificialSynthetic polypeptide 100Val Lys Lys Leu Leu Thr Leu Ser Ala Leu Ser Gln Trp His Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510128PRTartificialSynthetic polypeptide 101Val Lys Lys Leu Leu Ser Leu Val Tyr Phe Phe Pro Phe Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510228PRTartificialSynthetic polypeptide 102Val Lys Lys Leu Leu Phe Ala Phe Ala Pro Ala Pro Phe Tyr His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510328PRTartificialSynthetic polypeptide 103Val Lys Lys Leu Leu Phe Leu Pro Phe Ala Leu Val Pro Arg Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510428PRTartificialSynthetic polypeptide 104Val Lys Lys Leu Leu Ala Leu Trp Met Gln Leu Tyr Pro Gln Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510528PRTartificialSynthetic polypeptide 105Val Lys Lys Leu Leu Ala Ser Ile Leu Phe Ser His Ala Ala Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510628PRTartificialSynthetic polypeptide 106Val Lys Lys Leu Leu Leu Pro Leu Pro Trp Ser Leu His Leu Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510728PRTartificialSynthetic polypeptide 107Val Lys Lys Leu Leu Leu Pro His Phe Met Ser Phe Trp Phe Glu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510828PRTartificialSynthetic polypeptide 108Val Lys Lys Leu Leu Leu Phe Gln Pro Phe Trp Pro Ile Pro Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2510928PRTartificialSynthetic polypeptide 109Val Lys Lys Leu Leu Leu Leu Phe Ser Leu Gly Arg Leu Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511028PRTartificialSynthetic polypeptide 110Val Lys Lys Leu Leu Pro Leu Trp Val Leu Leu Lys Asp Pro Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511128PRTartificialSynthetic polypeptide 111Val Lys Lys Leu Leu Met Ser Phe Ala Thr Leu Phe Pro His Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511228PRTartificialSynthetic polypeptide 112Val Lys Lys Leu Leu Gln His Ser Leu Val Thr Ser Trp Leu Cys Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511328PRTartificialSynthetic polypeptide 113Val Lys Lys Leu Leu Leu Leu Phe Gln Gly Ala Phe Val Gly Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511428PRTartificialSynthetic polypeptide 114Val Lys Lys Leu Leu Trp Met Phe His Ser Leu Pro Phe Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511528PRTartificialSynthetic polypeptide 115Val Lys Lys Leu Leu Leu Thr Gln Leu Leu Leu Thr Arg Leu His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511628PRTartificialSynthetic polypeptide 116Val Lys Lys Leu Leu Ala Leu Thr Leu Val Pro Ser Ser Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511728PRTartificialSynthetic polypeptide 117Val Lys Lys Leu Leu Leu Pro Trp Tyr Met Leu Leu Ser Asp Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511828PRTartificialSynthetic polypeptide 118Val Lys Lys Leu Leu Val Val Thr Gln Phe Trp Pro Ser Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2511928PRTartificialSynthetic polypeptide 119Val Lys Lys Leu Leu Leu Ser Thr Leu Phe Leu Trp His Val Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512028PRTartificialSynthetic polypeptide 120Val Lys Lys Leu Leu Arg Ser Leu Phe Phe Gln Gln Leu Tyr Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512128PRTartificialSynthetic polypeptide 121Val Lys Lys Leu Leu Thr Leu Thr Thr Leu His Gln Thr Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512228PRTartificialSynthetic polypeptide 122Val Lys Lys Leu Leu Ser Ala Leu Leu Ala Pro Trp Tyr Trp Asp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512328PRTartificialSynthetic polypeptide 123Val Lys Lys Leu Leu Ala Ile Gln Gln Arg Met Gln Ile Tyr Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512428PRTartificialSynthetic polypeptide 124Val Lys Lys Leu Leu Leu Leu Phe Pro Trp Phe Gln Pro Pro Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512528PRTartificialSynthetic polypeptide 125Val Lys Lys Leu Leu Tyr Phe Thr Ser Leu Leu Gly Gln Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512628PRTartificialSynthetic polypeptide 126Val Lys Lys Leu Leu Pro Val Leu Ile Phe Leu Ser Glu Ile Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512728PRTartificialSynthetic polypeptide 127Val Lys Lys Leu Leu Val Ala Thr Ser Leu Arg Trp Ala Val Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512828PRTartificialSynthetic polypeptide 128Val Lys Lys Leu Leu Ala Gln Leu Phe His Leu Phe Ala Thr His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2512928PRTartificialSynthetic polypeptide 129Val Lys Lys Leu Leu Leu Gln Phe Ser Ala Leu Phe Asn Ser Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513028PRTartificialSynthetic polypeptide 130Val Lys Lys Leu Leu Phe His Leu Met Ser Met Leu Pro Pro Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513128PRTartificialSynthetic polypeptide 131Val Lys Lys Leu Leu Pro Val Cys Ser Gln Ser Met Phe Pro Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513228PRTartificialSynthetic polypeptide 132Val Lys Lys Leu Leu Leu Leu Leu Ser Ser Ser Tyr Gln Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513328PRTartificialSynthetic polypeptide 133Val Lys Lys Leu Leu Leu Asp Ser Leu Phe Phe His Ala Pro Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513428PRTartificialSynthetic polypeptide 134Val Lys Lys Leu Leu Gln Ala Trp Val Phe Ser Ala His Gln Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513528PRTartificialSynthetic polypeptide 135Val Lys Lys Leu Leu Phe Gln Ala Leu Gly Ala Leu Thr Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513628PRTartificialSynthetic polypeptide 136Val Lys Lys Leu Leu Cys Phe Phe Phe Phe Leu Gln Phe His Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513728PRTartificialSynthetic polypeptide 137Val Lys Lys Leu Leu Cys Phe Ser His Leu Ala Leu Pro Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513828PRTartificialSynthetic polypeptide 138Val Lys Lys Leu Leu Phe Gly Ser Trp Ile Pro Phe Thr Gln Met Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2513928PRTartificialSynthetic polypeptide 139Val Lys Lys Leu Leu Gly Leu Gly Tyr Phe Asn Trp Thr Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514028PRTartificialSynthetic polypeptide 140Val Lys Lys Leu Leu His Leu Phe Pro Leu Phe Gln Phe His His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514128PRTartificialSynthetic polypeptide 141Val Lys Lys Leu Leu Ser Glu His Val Ser Ser Ile Cys Val Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514228PRTartificialSynthetic polypeptide 142Val Lys Lys Leu Leu Phe Ser Cys Leu Leu Asp Pro Thr Cys Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514328PRTartificialSynthetic polypeptide 143Val Lys Lys Leu Leu Leu Tyr Leu Leu His Pro Ser Phe Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514428PRTartificialSynthetic polypeptide 144Val Lys Lys Leu Leu Trp Cys

Ala Pro Leu Leu Tyr Ser Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514528PRTartificialSynthetic polypeptide 145Val Lys Lys Leu Leu Phe Ala Met Phe Pro Tyr Thr Phe Gln Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514628PRTartificialSynthetic polypeptide 146Val Lys Lys Leu Leu Leu Pro Ser Leu Phe Tyr Val Glu Ser Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514728PRTartificialSynthetic polypeptide 147Val Lys Lys Leu Leu Ser Leu Trp Leu Ser Ser Leu Ser Val Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514828PRTartificialSynthetic polypeptide 148Val Lys Lys Leu Leu Pro His Leu Trp Phe Leu Trp Ser Leu Lys Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2514928PRTartificialSynthetic polypeptide 149Val Lys Lys Leu Leu Ala Ser Asp Pro Val Trp Tyr Phe Leu Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515028PRTartificialSynthetic polypeptide 150Val Lys Lys Leu Leu Gly Leu Pro Leu Met Gly Leu Gln Ser Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515128PRTartificialSynthetic polypeptide 151Val Lys Lys Leu Leu Pro Gln Leu Leu Leu Leu Arg Ala Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515228PRTartificialSynthetic polypeptide 152Val Lys Lys Leu Leu Ala Pro Ser Ala Phe Ser Leu His Leu Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515328PRTartificialSynthetic polypeptide 153Val Lys Lys Leu Leu Phe Gln Leu Ser Ser Leu Phe Val Pro Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515428PRTartificialSynthetic polypeptide 154Val Lys Lys Leu Leu Val Pro Ser Phe Leu Ser Thr Met Ile Glu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515528PRTartificialSynthetic polypeptide 155Val Lys Lys Leu Leu Ala Ser Pro Phe Phe Ala Ser Tyr Leu Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515628PRTartificialSynthetic polypeptide 156Val Lys Lys Leu Leu Leu Gln Tyr Leu Leu Ser Pro Ile Gly Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515728PRTartificialSynthetic polypeptide 157Val Lys Lys Leu Leu Val Leu Ser Val Pro Ile Ser Ala His His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515828PRTartificialSynthetic polypeptide 158Val Lys Lys Leu Leu Met Met Gln Ala Leu Ser Ser Leu Pro Glu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2515928PRTartificialSynthetic polypeptide 159Val Lys Lys Leu Leu Met Pro Ala Val Leu Ala Thr Arg Leu Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516028PRTartificialSynthetic polypeptide 160Val Lys Lys Leu Leu Pro Phe Thr Ala Trp Ile Ile Asp Gly Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516128PRTartificialSynthetic polypeptide 161Val Lys Lys Leu Leu Thr Gln Leu Leu Pro Leu Trp Gln Pro Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516228PRTartificialSynthetic polypeptide 162Val Lys Lys Leu Leu Leu Val Pro Ser Leu Leu Pro Leu Thr Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516328PRTartificialSynthetic polypeptide 163Val Lys Lys Leu Leu Pro Ile Gln Ser Cys Met Val Ile Pro Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516428PRTartificialSynthetic polypeptide 164Val Lys Lys Leu Leu Trp Ser Leu His Leu Ala Thr Arg Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516528PRTartificialSynthetic polypeptide 165Val Lys Lys Leu Leu Gln Gln Val Leu Leu Cys Ser Thr Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516628PRTartificialSynthetic polypeptide 166Val Lys Lys Leu Leu Leu Leu Arg Tyr Phe Leu Asp Pro Met Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516728PRTartificialSynthetic polypeptide 167Val Lys Lys Leu Leu Ile Pro Gln Phe Leu Arg Ser His His Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516828PRTartificialSynthetic polypeptide 168Val Lys Lys Leu Leu Gly Val Leu His Leu Ala Leu Ser Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2516928PRTartificialSynthetic polypeptide 169Val Lys Lys Leu Leu Leu Val Thr Ser Gln Phe Ser Leu Val Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517028PRTartificialSynthetic polypeptide 170Val Lys Lys Leu Leu Pro Leu Ala Leu Ser Trp Phe Gln Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517128PRTartificialSynthetic polypeptide 171Val Lys Lys Leu Leu Gln His Gln Trp Tyr Pro Thr Val Leu Met Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517228PRTartificialSynthetic polypeptide 172Val Lys Lys Leu Leu Leu Met Tyr Trp Leu Ser Lys Pro Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517328PRTartificialSynthetic polypeptide 173Val Lys Lys Leu Leu Thr Gln Leu Thr Leu Ser Ser Ser Pro Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517428PRTartificialSynthetic polypeptide 174Val Lys Lys Leu Leu Gln Leu Thr Ala Leu Leu Ser Arg Leu Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517528PRTartificialSynthetic polypeptide 175Val Lys Lys Leu Leu Leu Met Thr Phe Gly Thr Thr Pro Gln Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517628PRTartificialSynthetic polypeptide 176Val Lys Lys Leu Leu Ser Ala Phe Ser Phe Ser Leu Ser Ser Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517728PRTartificialSynthetic polypeptide 177Val Lys Lys Leu Leu Ala Pro Trp Leu Val Leu Pro His Phe Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517828PRTartificialSynthetic polypeptide 178Val Lys Lys Leu Leu His Val Leu Ser Phe Ala Pro Pro Met Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2517928PRTartificialSynthetic polypeptide 179Val Lys Lys Leu Leu Asn Trp Leu Phe Phe Ala His Pro Phe Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518028PRTartificialSynthetic polypeptide 180Val Lys Lys Leu Leu Gln Leu Ala Val Leu Leu Gly Ser Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518128PRTartificialSynthetic polypeptide 181Val Lys Lys Leu Leu Leu Phe Gly Leu Phe Tyr Phe Arg Ala Cys Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518228PRTartificialSynthetic polypeptide 182Val Lys Lys Leu Leu Phe Gln Phe Phe Val Val Trp Arg Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518328PRTartificialSynthetic polypeptide 183Val Lys Lys Leu Leu Pro Trp Ala Trp Pro Pro Pro Pro Phe Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518428PRTartificialSynthetic polypeptide 184Val Lys Lys Leu Leu Leu Gln Leu Val Ile Val Tyr Tyr Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518528PRTartificialSynthetic polypeptide 185Val Lys Lys Leu Leu Arg Gln Ser Val Leu Leu Ser Ala Leu His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518628PRTartificialSynthetic polypeptide 186Val Lys Lys Leu Leu Val Tyr Gly Tyr Phe Leu Thr Thr Phe Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518728PRTartificialSynthetic polypeptide 187Val Lys Lys Leu Leu Cys Phe Ser Pro Leu Phe Gly Phe His Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518828PRTartificialSynthetic polypeptide 188Val Lys Lys Leu Leu Pro Gly Tyr Ala Leu Trp Gln Thr Ile Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2518928PRTartificialSynthetic polypeptide 189Val Lys Lys Leu Leu Gln Arg Ile Phe Ile Cys Phe Phe Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519028PRTartificialSynthetic polypeptide 190Val Lys Lys Leu Leu Pro His Val Phe Ser Cys Gln Leu Ser Ala Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519128PRTartificialSynthetic polypeptide 191Val Lys Lys Leu Leu Ser Pro Leu Ser Leu Ser Val Lys Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519228PRTartificialSynthetic polypeptide 192Val Lys Lys Leu Leu Ala Arg Ser Leu Phe Ser Gly Ser Met Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519328PRTartificialSynthetic polypeptide 193Val Lys Lys Leu Leu Leu Gln Phe Leu Ile Val Phe Pro Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519428PRTartificialSynthetic polypeptide 194Val Lys Lys Leu Leu Leu Ala Val Leu Leu Gly Gln Ser Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519528PRTartificialSynthetic polypeptide 195Val Lys Lys Leu Leu Leu Leu Ser His Leu Phe Leu Arg Leu His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519628PRTartificialSynthetic polypeptide 196Val Lys Lys Leu Leu Leu Ala Met Val Phe Phe Val Thr Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519728PRTartificialSynthetic polypeptide 197Val Lys Lys Leu Leu Trp Leu Phe Ala Leu Pro Gln Glu Asn Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519828PRTartificialSynthetic polypeptide 198Val Lys Lys Leu Leu His Pro Leu Val Leu Leu Ser Ser Ser Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2519928PRTartificialSynthetic polypeptide 199Val Lys Lys Leu Leu Leu Gln Tyr Leu Phe Met Leu Ser Met Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520028PRTartificialSynthetic polypeptide 200Val Lys Lys Leu Leu Pro Ala Leu Leu Ile Arg Tyr Ala Ser Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520128PRTartificialSynthetic polypeptide 201Val Lys Lys Leu Leu Gln Gln Phe Thr Ser Pro Phe Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520228PRTartificialSynthetic polypeptide 202Val Lys Lys Leu Leu Ser Pro Cys Phe Phe Leu Leu Tyr Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520328PRTartificialSynthetic polypeptide 203Val Lys Lys Leu Leu Pro Gly Met Pro Leu Phe Phe Thr Asn Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520428PRTartificialSynthetic polypeptide 204Val Lys Lys Leu Leu Pro Gln Val Phe Phe Leu Phe Arg Pro Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520528PRTartificialSynthetic polypeptide 205Val Lys Lys Leu Leu Pro Phe Pro Ile Leu Leu Gln Ser Pro Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520628PRTartificialSynthetic polypeptide 206Val Lys Lys Leu Leu Phe Gln Ala Cys Cys Leu Phe Pro Leu Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520728PRTartificialSynthetic polypeptide 207Val Lys Lys Leu Leu Ala Val Val His Thr Met Pro Leu Phe Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520828PRTartificialSynthetic polypeptide 208Val Lys Lys Leu Leu Gln Phe Ser Trp Ala Phe Val Ser Ile Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2520928PRTartificialSynthetic polypeptide 209Val Lys Lys Leu Leu Pro Val Cys Leu Phe Trp Ser Phe Phe Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521028PRTartificialSynthetic polypeptide 210Val Lys Lys Leu Leu Gln Leu Leu Trp Gln Gln Gln Val Pro Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521128PRTartificialSynthetic polypeptide 211Val Lys Lys Leu Leu Pro Leu Gln Ala Leu Ser Trp Phe Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521228PRTartificialSynthetic polypeptide 212Val Lys Lys Leu Leu Phe Tyr Leu Leu Cys Arg Leu Ser Leu Gln Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521328PRTartificialSynthetic polypeptide 213Val Lys Lys Leu Leu Tyr Leu Gln Ile Leu Val Ile Cys Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521428PRTartificialSynthetic polypeptide 214Val Lys Lys Leu Leu Gln Leu Phe Leu Ile Val Phe Pro Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521528PRTartificialSynthetic polypeptide 215Val Lys Lys Leu Leu Pro Leu His Phe Ala Leu Phe Phe Arg Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20

2521628PRTartificialSynthetic polypeptide 216Val Lys Lys Leu Leu Pro Phe Pro Met His Leu Val Leu Pro Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521728PRTartificialSynthetic polypeptide 217Val Lys Lys Leu Leu Pro Leu Leu Phe Ser Pro Pro Ser Leu His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521828PRTartificialSynthetic polypeptide 218Val Lys Lys Leu Leu Cys Gln Ser Ile Thr Phe Ser Ser Ile Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2521928PRTartificialSynthetic polypeptide 219Val Lys Lys Leu Leu Trp Gln Arg Leu Phe Pro Phe Leu Leu Ile Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522028PRTartificialSynthetic polypeptide 220Val Lys Lys Leu Leu Met Val Pro Phe Trp Pro Phe Ser Phe Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522128PRTartificialSynthetic polypeptide 221Val Lys Lys Leu Leu Gln Ala Phe Pro Leu Pro Pro Leu Leu Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522228PRTartificialSynthetic polypeptide 222Val Lys Lys Leu Leu Pro Leu Tyr Leu Leu Phe Arg Ser Phe Val Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522328PRTartificialSynthetic polypeptide 223Val Lys Lys Leu Leu His Arg Ser Met Tyr Leu Ser Trp Leu Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522428PRTartificialSynthetic polypeptide 224Val Lys Lys Leu Leu Leu Leu Ser Thr Leu Val Arg Ala Pro Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522528PRTartificialSynthetic polypeptide 225Val Lys Lys Leu Leu Pro Leu Ala Leu Ser Gln Trp Phe Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522628PRTartificialSynthetic polypeptide 226Val Lys Lys Leu Leu Ala Gln Gly Met Ile Phe Phe Leu Arg Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522728PRTartificialSynthetic polypeptide 227Val Lys Lys Leu Leu Phe Cys Cys Arg Leu Ala Leu Gln Phe Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522828PRTartificialSynthetic polypeptide 228Val Lys Lys Leu Leu Tyr Leu Gln Phe Leu Ser Leu Met Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2522928PRTartificialSynthetic polypeptide 229Val Lys Lys Leu Leu Cys Gln Ala Thr Phe Pro Thr Leu Leu Cys Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523028PRTartificialSynthetic polypeptide 230Val Lys Lys Leu Leu Ala Arg Ser Tyr Leu Tyr Phe Ser Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523128PRTartificialSynthetic polypeptide 231Val Lys Lys Leu Leu Tyr Gln Ser Ser Phe Leu Pro Leu Phe Trp Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523228PRTartificialSynthetic polypeptide 232Val Lys Lys Leu Leu Ser Ala Ser Phe Leu Ala Phe Arg Ile Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523328PRTartificialSynthetic polypeptide 233Val Lys Lys Leu Leu Ser Val Leu Phe Leu Ser His Tyr His Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523428PRTartificialSynthetic polypeptide 234Val Lys Lys Leu Leu Pro Leu Ala Leu Leu Tyr Val Arg Leu Ser Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523528PRTartificialSynthetic polypeptide 235Val Lys Lys Leu Leu Pro Glu Phe Leu Leu Leu Phe Arg Phe Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523628PRTartificialSynthetic polypeptide 236Val Lys Lys Leu Leu Phe Pro Ser Leu Tyr Ala Trp Gly Gly Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523728PRTartificialSynthetic polypeptide 237Val Lys Lys Leu Leu Leu Gln Ala Ala Ala Phe Phe Cys Trp Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523828PRTartificialSynthetic polypeptide 238Val Lys Lys Leu Leu Pro Phe Phe Leu Phe Cys Ser Ser Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2523928PRTartificialSynthetic polypeptide 239Val Lys Lys Leu Leu Glu Leu Thr Gln Leu Trp Leu Phe His Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524028PRTartificialSynthetic polypeptide 240Val Lys Lys Leu Leu Pro Gly Val Pro Leu Leu Leu Cys Phe Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524128PRTartificialSynthetic polypeptide 241Val Lys Lys Leu Leu Ser Gln Ala Tyr Leu Ser Tyr Phe Leu Tyr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524228PRTartificialSynthetic polypeptide 242Val Lys Lys Leu Leu Ile Ser Tyr Ala Phe Leu Val Arg Val Thr Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524328PRTartificialSynthetic polypeptide 243Val Lys Lys Leu Leu Ala Pro Ala Leu Leu Arg Ser Ile Leu Ala Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524428PRTartificialSynthetic polypeptide 244Val Lys Lys Leu Leu His Ser His Thr Leu Leu Met Ser Leu His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524528PRTartificialSynthetic polypeptide 245Val Lys Lys Leu Leu Ala Val Ser Ala Phe Val Ser Leu Val Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524628PRTartificialSynthetic polypeptide 246Val Lys Lys Leu Leu Thr Leu Ile Thr Phe Lys Phe Leu Pro His Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524728PRTartificialSynthetic polypeptide 247Val Lys Lys Leu Leu Gln Gln Phe Ala Ile Pro Leu Val Glu Phe Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524828PRTartificialSynthetic polypeptide 248Val Lys Lys Leu Leu Met Pro Cys Leu Leu Val Tyr Tyr Leu Glu Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2524928PRTartificialSynthetic polypeptide 249Val Lys Lys Leu Leu Arg Tyr Cys Leu Leu Leu Gln Ile Val Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525028PRTartificialSynthetic polypeptide 250Val Lys Lys Leu Leu Ser Leu Ala Leu Leu Arg Val Ser Leu Gly Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525128PRTartificialSynthetic polypeptide 251Val Lys Lys Leu Leu Ile Ile Gly Arg Ile Ala Leu Ile Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525228PRTartificialSynthetic polypeptide 252Val Lys Lys Leu Leu Pro Gln Leu Ile Cys Ala Phe Ile Leu Arg Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525328PRTartificialSynthetic polypeptide 253Val Lys Lys Leu Leu Met Val Pro Leu Phe Pro Leu Pro Leu Pro Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525428PRTartificialSynthetic polypeptide 254Val Lys Lys Leu Leu His Gln Ala Ile Leu Tyr Tyr Tyr Leu Asn Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His 20 2525528PRTartificialSynthetic polypeptide 255Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Pro Ala Gln Ala Met1 5 10 15Pro Met Ser Arg Met Ala His His His His His His 20 2525628PRTartificialSynthetic polypeptide 256Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Val Leu Val Arg1 5 10 15Glu Ser Ser Ser Met Ala His His His His His His 20 2525728PRTartificialSynthetic polypeptide 257Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Leu Val Val Ser Ser1 5 10 15Arg Thr Arg Ala Met Ala His His His His His His 20 2525828PRTartificialSynthetic polypeptide 258Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Leu Ser Arg Pro Arg1 5 10 15Ala Val Pro Asp Met Ala His His His His His His 20 2525928PRTartificialSynthetic polypeptide 259Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Cys Val Ser Val Arg Ser1 5 10 15Pro Ala Phe Ala Met Ala His His His His His His 20 2526028PRTartificialSynthetic polypeptide 260Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Met Thr Thr Leu Ala Ser1 5 10 15Arg Thr His Ala Met Ala His His His His His His 20 2526128PRTartificialSynthetic polypeptide 261Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ser Met Thr Arg1 5 10 15Ser Gly Ala Ala Met Ala His His His His His His 20 2526228PRTartificialSynthetic polypeptide 262Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Leu Arg Ser Ser Val1 5 10 15Pro Val Asp Ser Met Ala His His His His His His 20 2526328PRTartificialSynthetic polypeptide 263Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ser Ser Leu Thr Arg1 5 10 15Asp Ser Ser Ser Met Ala His His His His His His 20 2526428PRTartificialSynthetic polypeptide 264Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Gly Leu Phe Thr Ile Arg1 5 10 15Asp Ser Phe Ala Met Ala His His His His His His 20 2526528PRTartificialSynthetic polypeptide 265Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Leu Gly Ile Thr Lys1 5 10 15Pro Val Trp Ser Met Ala His His His His His His 20 2526628PRTartificialSynthetic polypeptide 266Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Thr Leu Thr Pro Arg1 5 10 15Pro Val Phe Ser Met Ala His His His His His His 20 2526728PRTartificialSynthetic polypeptide 267Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Gln Leu Ala Leu Ser Arg1 5 10 15Pro Ser Phe Pro Met Ala His His His His His His 20 2526828PRTartificialSynthetic polypeptide 268Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Ser Phe Leu Val Ala1 5 10 15Asp Gln Ser Ser Met Ala His His His His His His 20 2526928PRTartificialSynthetic polypeptide 269Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Leu Gly Leu Ala Ser1 5 10 15Pro Arg Ser Arg Met Ala His His His His His His 20 2527028PRTartificialSynthetic polypeptide 270Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Thr Leu Ser Asn Arg1 5 10 15Ser Ala Trp Ser Met Ala His His His His His His 20 2527128PRTartificialSynthetic polypeptide 271Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ser Leu Tyr Pro Thr1 5 10 15Arg Ser Thr Ala Met Ala His His His His His His 20 2527228PRTartificialSynthetic polypeptide 272Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Thr Thr Leu Ser Arg1 5 10 15Pro Ser Phe Ser Met Ala His His His His His His 20 2527328PRTartificialSynthetic polypeptide 273Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Ser Arg Pro Pro1 5 10 15Gln Pro Ser Ser Met Ala His His His His His His 20 2527428PRTartificialSynthetic polypeptide 274Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Thr Met Ser Ser Pro Pro1 5 10 15Arg Ser Thr Ser Met Ala His His His His His His 20 2527528PRTartificialSynthetic polypeptide 275Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Leu Arg Ile Ser1 5 10 15Pro Ser Ala Ser Met Ala His His His His His His 20 2527628PRTartificialSynthetic polypeptide 276Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Phe Leu Arg Pro Ser1 5 10 15Ala Ala Arg Pro Met Ala His His His His His His 20 2527728PRTartificialSynthetic polypeptide 277Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Trp Ser Ser Ser Arg1 5 10 15Pro Thr Ser Gln Met Ala His His His His His His 20 2527828PRTartificialSynthetic polypeptide 278Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Val Cys Ser Arg1 5 10 15Pro Leu His Ala Met Ala His His His His His His 20 2527928PRTartificialSynthetic polypeptide 279Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Leu Gln Arg Pro Pro1 5 10 15Ser Pro Asn Thr Met Ala His His His His His His 20 2528028PRTartificialSynthetic polypeptide 280Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ala Met Ala Ser Phe Arg1 5 10 15Pro Arg Asp Gln Met Ala His His His His His His 20 2528128PRTartificialSynthetic polypeptide 281Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Arg Ser Leu Ala Met1 5 10 15Gln Pro Leu Pro Met Ala His His His His His His 20 2528228PRTartificialSynthetic polypeptide 282Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ser Ser Leu Arg Ser1 5 10 15Ser Asn Pro Glu Met Ala His His His His His His 20 2528328PRTartificialSynthetic polypeptide 283Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Ile Leu Ile Asn Phe1 5 10 15Arg Ala Ser Ser Met Ala His His His His His His 20 2528428PRTartificialSynthetic polypeptide 284Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Trp Arg Ser Phe Trp1 5 10 15Glu Pro Pro Ala Met Ala His His His His His His 20 2528528PRTartificialSynthetic polypeptide 285Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ala Ala Pro Arg1 5 10 15Ser Thr Val Ala Met Ala His His His His His His 20 2528628PRTartificialSynthetic polypeptide 286Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Gln Tyr Ser Ala Phe Ser1 5 10 15Met Ser Pro Arg Met Ala His His His His His His 20 2528728PRTartificialSynthetic polypeptide 287Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Val Ser Ser Lys1 5 10 15Asn Ser Tyr Pro Met Ala His

His His His His His 20 2528828PRTartificialSynthetic polypeptide 288Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Gly Leu Ser Val Ser Phe1 5 10 15Arg Thr Ser Ala Met Ala His His His His His His 20 2528928PRTartificialSynthetic polypeptide 289Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ala Met Leu Glu Pro Thr1 5 10 15Arg Ser Ser Ala Met Ala His His His His His His 20 2529028PRTartificialSynthetic polypeptide 290Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Leu Ser Leu His Arg1 5 10 15Pro Ala Leu Ala Met Ala His His His His His His 20 2529128PRTartificialSynthetic polypeptide 291Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ser Ala Ser Ala Arg1 5 10 15Gly Ser Tyr Ala Met Ala His His His His His His 20 2529228PRTartificialSynthetic polypeptide 292Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ala Val Thr His1 5 10 15Arg Ala Tyr Ser Met Ala His His His His His His 20 2529328PRTartificialSynthetic polypeptide 293Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Phe Ser Leu Ser Arg1 5 10 15Tyr Ser Leu Ala Met Ala His His His His His His 20 2529428PRTartificialSynthetic polypeptide 294Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ser Ala Pro Arg1 5 10 15His Ala Ser Pro Met Ala His His His His His His 20 2529528PRTartificialSynthetic polypeptide 295Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Ser Phe Ser Arg Leu1 5 10 15Pro Ser Ser Asp Met Ala His His His His His His 20 2529628PRTartificialSynthetic polypeptide 296Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ser Leu Thr Lys1 5 10 15Pro Ser Leu Ser Met Ala His His His His His His 20 2529728PRTartificialSynthetic polypeptide 297Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Ser Pro Ala Thr Glu1 5 10 15Val Leu Ser Pro Met Ala His His His His His His 20 2529828PRTartificialSynthetic polypeptide 298Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Thr Leu Phe Leu Gln Arg1 5 10 15Ser Ser Leu Ala Met Ala His His His His His His 20 2529928PRTartificialSynthetic polypeptide 299Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Phe Thr Arg Val Pro1 5 10 15His Lys Pro Ser Met Ala His His His His His His 20 2530028PRTartificialSynthetic polypeptide 300Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ala Ile Thr Arg Ser Ser1 5 10 15Gln Phe Pro Ser Met Ala His His His His His His 20 2530128PRTartificialSynthetic polypeptide 301Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Gly Asp Leu Arg Ser1 5 10 15Ser Pro Asp Ala Met Ala His His His His His His 20 2530228PRTartificialSynthetic polypeptide 302Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Thr Thr Leu Ser Thr1 5 10 15Arg Cys Tyr Ala Met Ala His His His His His His 20 2530328PRTartificialSynthetic polypeptide 303Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Asp Ala Ser Leu Glu1 5 10 15Gly Pro Ala Met Met Ala His His His His His His 20 2530428PRTartificialSynthetic polypeptide 304Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Ser Ser Pro Ser1 5 10 15Ser Arg Ala Pro Met Ala His His His His His His 20 2530528PRTartificialSynthetic polypeptide 305Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Phe Ser Phe Pro Phe1 5 10 15Arg Ser Ala Ala Met Ala His His His His His His 20 2530628PRTartificialSynthetic polypeptide 306Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ser Met Ser Ser1 5 10 15Pro Ala Arg Ser Met Ala His His His His His His 20 2530728PRTartificialSynthetic polypeptide 307Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Trp Ser Leu Cys Arg1 5 10 15Pro Val Cys Ala Met Ala His His His His His His 20 2530828PRTartificialSynthetic polypeptide 308Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Tyr Cys Trp Pro Arg1 5 10 15His Ser Trp Ser Met Ala His His His His His His 20 2530928PRTartificialSynthetic polypeptide 309Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ile Phe Tyr Thr Thr Arg1 5 10 15Ser Ser Leu Ser Met Ala His His His His His His 20 2531028PRTartificialSynthetic polypeptide 310Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ile Tyr Thr Leu Arg Ser1 5 10 15His Ser Met Thr Met Ala His His His His His His 20 2531128PRTartificialSynthetic polypeptide 311Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Pro Val Pro Ser Leu Leu1 5 10 15Gly Ser Ala Asp Met Ala His His His His His His 20 2531228PRTartificialSynthetic polypeptide 312Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Leu Ser Leu Asn Ser1 5 10 15Arg Ser Tyr Pro Met Ala His His His His His His 20 2531328PRTartificialSynthetic polypeptide 313Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Ser Pro Thr Ser Gln1 5 10 15Glu Ile Arg His Met Ala His His His His His His 20 2531428PRTartificialSynthetic polypeptide 314Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Ser Cys Pro Leu1 5 10 15Arg Val Ala Ser Met Ala His His His His His His 20 2531528PRTartificialSynthetic polypeptide 315Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Leu Ser Leu Asn Arg1 5 10 15Gly Val Phe Ala Met Ala His His His His His His 20 2531628PRTartificialSynthetic polypeptide 316Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Pro Gln Val Leu Ser1 5 10 15Ser Ser Pro Gly Met Ala His His His His His His 20 2531728PRTartificialSynthetic polypeptide 317Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Val Asn Ala Met Ser1 5 10 15Ser Pro Arg Pro Met Ala His His His His His His 20 2531828PRTartificialSynthetic polypeptide 318Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Thr Phe Val Arg1 5 10 15Ser Ser Trp Cys Met Ala His His His His His His 20 2531928PRTartificialSynthetic polypeptide 319Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Asp Leu Ser Ser Asp1 5 10 15Ser Val Ser Pro Met Ala His His His His His His 20 2532028PRTartificialSynthetic polypeptide 320Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Ile Leu Phe Trp Arg1 5 10 15Asn Thr His Ala Met Ala His His His His His His 20 2532128PRTartificialSynthetic polypeptide 321Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Cys Phe Leu Ser Arg1 5 10 15Ser Ala Phe Ser Met Ala His His His His His His 20 2532228PRTartificialSynthetic polypeptide 322Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Phe Met Ile Thr Ser1 5 10 15Lys Ser Arg Ser Met Ala His His His His His His 20 2532328PRTartificialSynthetic polypeptide 323Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ile Val Ser Ser Ser Arg1 5 10 15Gly Ser Phe Ala Met Ala His His His His His His 20 2532428PRTartificialSynthetic polypeptide 324Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ala Ala Ser Arg Pro Leu1 5 10 15Ser Pro Ala Ala Met Ala His His His His His His 20 2532528PRTartificialSynthetic polypeptide 325Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Leu Phe Ser Pro Leu1 5 10 15Arg Ser Tyr Ser Met Ala His His His His His His 20 2532628PRTartificialSynthetic polypeptide 326Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Leu Ser Tyr Val Arg1 5 10 15Pro Leu Ser Ala Met Ala His His His His His His 20 2532728PRTartificialSynthetic polypeptide 327Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Ile Phe Thr Pro Arg1 5 10 15Ser Val His Ser Met Ala His His His His His His 20 2532828PRTartificialSynthetic polypeptide 328Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Ser Ser Ile Tyr Lys1 5 10 15Asn Ser Pro Pro Met Ala His His His His His His 20 2532928PRTartificialSynthetic polypeptide 329Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Met Ser Asp Ser Thr Ala1 5 10 15Pro Ser Phe Ala Met Ala His His His His His His 20 2533028PRTartificialSynthetic polypeptide 330Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Thr Leu Pro Gln Pro Arg1 5 10 15Phe Pro Ser Pro Met Ala His His His His His His 20 2533128PRTartificialSynthetic polypeptide 331Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Leu Leu Ala Asp Ser1 5 10 15Pro Arg Arg Pro Met Ala His His His His His His 20 2533228PRTartificialSynthetic polypeptide 332Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Thr Asp Asn Ser Gly1 5 10 15Glu Pro Ser Leu Met Ala His His His His His His 20 2533328PRTartificialSynthetic polypeptide 333Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Cys Met Pro Met Ser1 5 10 15Arg Thr Cys Ala Met Ala His His His His His His 20 2533428PRTartificialSynthetic polypeptide 334Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Met Ser Arg Leu Ser Tyr1 5 10 15His Thr Pro Ser Met Ala His His His His His His 20 2533528PRTartificialSynthetic polypeptide 335Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ser Asn Ser Arg Val1 5 10 15Pro Pro Ser Ser Met Ala His His His His His His 20 2533628PRTartificialSynthetic polypeptide 336Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Phe Ala Ser Met Arg1 5 10 15His Thr Gln Ala Met Ala His His His His His His 20 2533728PRTartificialSynthetic polypeptide 337Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Leu Ser Thr Ile Lys1 5 10 15Thr Ser Phe Ser Met Ala His His His His His His 20 2533828PRTartificialSynthetic polypeptide 338Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Gln Gln Ser Ser Leu1 5 10 15Ser Ser Val Pro Met Ala His His His His His His 20 2533928PRTartificialSynthetic polypeptide 339Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Thr Leu Ile Leu Ser His1 5 10 15Arg Ser Ser Ala Met Ala His His His His His His 20 2534028PRTartificialSynthetic polypeptide 340Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Phe Ser Arg Asp Pro1 5 10 15Ser Phe Thr Ser Met Ala His His His His His His 20 2534128PRTartificialSynthetic polypeptide 341Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ala Leu Ser Pro Thr Arg1 5 10 15His Thr Leu Ala Met Ala His His His His His His 20 2534228PRTartificialSynthetic polypeptide 342Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Asn Ile Leu Phe Thr Val1 5 10 15Arg Val Tyr Ala Met Ala His His His His His His 20 2534328PRTartificialSynthetic polypeptide 343Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ala Ser Leu Ser Ala1 5 10 15Arg Cys His Gly Met Ala His His His His His His 20 2534428PRTartificialSynthetic polypeptide 344Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Val Thr Leu Ser Leu1 5 10 15Arg Ala Ser Ala Met Ala His His His His His His 20 2534528PRTartificialSynthetic polypeptide 345Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser His Asp Pro Leu Leu1 5 10 15Leu Ser Ser Pro Met Ala His His His His His His 20 2534628PRTartificialSynthetic polypeptide 346Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Trp Ser Leu Ser Ser1 5 10 15Arg Gly Met Thr Met Ala His His His His His His 20 2534728PRTartificialSynthetic polypeptide 347Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ile Ser Tyr Cys Arg1 5 10 15Pro Val Ser Ser Met Ala His His His His His His 20 2534828PRTartificialSynthetic polypeptide 348Val Lys Lys Leu Leu Phe Ala Ile Pro Leu His Ser Val Glu Leu Pro1 5 10 15Ala Ser Pro Ala Met Ala His His His His His His 20 2534928PRTartificialSynthetic polypeptide 349Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Leu Ser Thr Ser Arg1 5 10 15Ser Ser Ser Gly Met Ala His His His His His His 20 2535028PRTartificialSynthetic polypeptide 350Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Phe Ser Cys Ser Arg1 5 10 15Phe Ala Leu Ser Met Ala His His His His His His 20 2535128PRTartificialSynthetic polypeptide 351Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Cys Thr Leu Ser Ser1 5 10 15Arg Ala Phe Ser Met Ala His His His His His His 20 2535228PRTartificialSynthetic polypeptide 352Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Ser Pro Leu Ala Arg1 5 10 15Asn Pro Phe Ser Met Ala His His His His His His 20 2535328PRTartificialSynthetic polypeptide 353Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Phe Ala Phe Ser Arg1 5 10 15Gln Ser Ser Gly Met Ala His His His His His His 20 2535428PRTartificialSynthetic polypeptide 354Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Thr Phe Ser Ile Phe Ser1 5 10 15Arg Ala Leu Ala Met Ala His His His His His His 20 2535528PRTartificialSynthetic polypeptide 355Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Leu Phe Phe Ser Ala1 5 10 15Arg Ala Ile Ala Met Ala His His His His His His 20 2535628PRTartificialSynthetic polypeptide 356Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Gln Pro Ser Leu Cys1 5 10 15Asp Pro Val Pro Met Ala His His His His His His 20 2535728PRTartificialSynthetic polypeptide 357Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Ala Ser Tyr His Arg1 5 10 15Val Ala Phe Ala Met Ala His His His His His His 20 2535828PRTartificialSynthetic polypeptide 358Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Gln Leu Trp Gln Leu1 5 10 15Pro Ser Arg Pro Met Ala His His His His His His 20 2535928PRTartificialSynthetic polypeptide 359Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Thr Pro Met Tyr Arg1

5 10 15Pro Thr Ser Pro Met Ala His His His His His His 20 2536028PRTartificialSynthetic polypeptide 360Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Leu Leu Ser Leu His Arg1 5 10 15Phe Ser Phe Ala Met Ala His His His His His His 20 2536128PRTartificialSynthetic polypeptide 361Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Tyr Ser His Pro Gln1 5 10 15Asn Ala Leu Ala Met Ala His His His His His His 20 2536228PRTartificialSynthetic polypeptide 362Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Val Leu Arg Ser Asp1 5 10 15Ala Ser Trp Gly Met Ala His His His His His His 20 2536328PRTartificialSynthetic polypeptide 363Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Ser Gly Pro Pro Phe1 5 10 15Asp Arg Thr Ser Met Ala His His His His His His 20 2536428PRTartificialSynthetic polypeptide 364Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Cys Ala Leu Ser Arg1 5 10 15Phe Thr His Ala Met Ala His His His His His His 20 2536528PRTartificialSynthetic polypeptide 365Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Phe Ser Leu Ser Arg Pro1 5 10 15Val Pro Pro Leu Met Ala His His His His His His 20 2536628PRTartificialSynthetic polypeptide 366Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Met Asp Ser Phe Ser1 5 10 15Arg Pro Phe Phe Met Ala His His His His His His 20 2536728PRTartificialSynthetic polypeptide 367Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Thr Ile Ile Pro Ser1 5 10 15Arg Ala Ser Ser Met Ala His His His His His His 20 2536828PRTartificialSynthetic polypeptide 368Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Pro Ser Ala Asn Pro1 5 10 15Pro Pro Leu Ser Met Ala His His His His His His 20 2536928PRTartificialSynthetic polypeptide 369Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Leu Ile Lys Pro Pro1 5 10 15Glu Gly Phe Ser Met Ala His His His His His His 20 2537028PRTartificialSynthetic polypeptide 370Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ile Ser Thr Leu His Phe1 5 10 15Arg Ala Phe Gly Met Ala His His His His His His 20 2537128PRTartificialSynthetic polypeptide 371Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Arg Val Met Cys Gly1 5 10 15His Ser Tyr Ala Met Ala His His His His His His 20 2537228PRTartificialSynthetic polypeptide 372Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Leu Ser Leu Ser Arg1 5 10 15Thr Phe Ser Gly Met Ala His His His His His His 20 2537328PRTartificialSynthetic polypeptide 373Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Trp Cys Ala Leu Ser Arg1 5 10 15Gln Ser Met Pro Met Ala His His His His His His 20 2537428PRTartificialSynthetic polypeptide 374Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Phe Trp Ser Leu Arg1 5 10 15Val Ser Trp Pro Met Ala His His His His His His 20 2537528PRTartificialSynthetic polypeptide 375Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Ile Leu Ser Pro Arg1 5 10 15Leu Pro Pro Pro Met Ala His His His His His His 20 2537628PRTartificialSynthetic polypeptide 376Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Ala Ala His Arg1 5 10 15Phe Ser Tyr Ala Met Ala His His His His His His 20 2537728PRTartificialSynthetic polypeptide 377Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Val His Leu Thr Ser1 5 10 15Lys Ala Ile Pro Met Ala His His His His His His 20 2537828PRTartificialSynthetic polypeptide 378Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Ser Leu Thr Leu Tyr Arg1 5 10 15Ser Gly Trp Ser Met Ala His His His His His His 20 2537928PRTartificialSynthetic polypeptide 379Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Tyr Tyr Ala Leu Ser Gly1 5 10 15Arg Pro Val Thr Met Ala His His His His His His 20 2538028PRTartificialSynthetic polypeptide 380Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Met Leu Ser Leu Met Arg1 5 10 15Gln Ser Ala Pro Met Ala His His His His His His 20 2538128PRTartificialSynthetic polypeptide 381Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Pro Leu Thr Arg Ile Gln Thr Pro His His His 20 2538228PRTartificialSynthetic polypeptide 382Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Thr Gln Leu Ser Arg Arg Glu Pro Ser His His His 20 2538328PRTartificialSynthetic polypeptide 383Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Ala Thr Ser Pro Ser Arg His His His 20 2538428PRTartificialSynthetic polypeptide 384Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Ala Arg Ser Tyr Met Leu Val Arg Pro His His His 20 2538528PRTartificialSynthetic polypeptide 385Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Tyr Met Leu Leu Ser Arg Pro His His His 20 2538628PRTartificialSynthetic polypeptide 386Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Ala Leu Ala Phe Phe Leu Pro His His His 20 2538728PRTartificialSynthetic polypeptide 387Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Gly Phe Thr Leu Pro Arg Leu Ile His His His 20 2538828PRTartificialSynthetic polypeptide 388Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Ala Phe Thr Arg Pro Ile Arg Pro His His His 20 2538928PRTartificialSynthetic polypeptide 389Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Tyr Ser His Ala Phe Met Leu Ile His His His 20 2539028PRTartificialSynthetic polypeptide 390Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Pro Met Ser Met Phe Arg Ser Asp His His His 20 2539128PRTartificialSynthetic polypeptide 391Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Ser Met Ser Gln Tyr Arg Gln Asn His His His 20 2539228PRTartificialSynthetic polypeptide 392Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Tyr Ser Arg Pro Pro Ser Ile His His His 20 2539328PRTartificialSynthetic polypeptide 393Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Ser Met Ser Arg Leu Arg Pro His His His His 20 2539428PRTartificialSynthetic polypeptide 394Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ser Leu Ser Arg Pro Met Leu Val His His His 20 2539528PRTartificialSynthetic polypeptide 395Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Ser Leu His Pro Thr Ala His His His 20 2539628PRTartificialSynthetic polypeptide 396Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Met Thr Arg Leu Ala Pro Pro His His His 20 2539728PRTartificialSynthetic polypeptide 397Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ala Met Ser Val Ser His Lys Thr His His His 20 2539828PRTartificialSynthetic polypeptide 398Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Leu Ala Pro Lys Pro Ser Val Lys Arg His His His 20 2539928PRTartificialSynthetic polypeptide 399Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Pro Ala Pro Ala Leu Ser Arg Leu His His His 20 2540028PRTartificialSynthetic polypeptide 400Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Lys Ala Met Ser Ala Arg Tyr Gln Ser His His His 20 2540128PRTartificialSynthetic polypeptide 401Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Ala Ser Gln Arg Ser Ser Pro Ile Arg His His His 20 2540228PRTartificialSynthetic polypeptide 402Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Leu Ser Phe Thr Ser Ala Arg Phe Gln His His His 20 2540328PRTartificialSynthetic polypeptide 403Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Ser Ala Ser Ser Arg Leu Ser Pro Lys His His His 20 2540428PRTartificialSynthetic polypeptide 404Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Tyr Thr Arg Val Pro Leu Ala His His His 20 2540528PRTartificialSynthetic polypeptide 405Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Thr Phe Leu Pro Pro Arg His His His 20 2540628PRTartificialSynthetic polypeptide 406Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Thr Arg Val Asn Ala Phe Met Leu Val His His His 20 2540728PRTartificialSynthetic polypeptide 407Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Ala Phe Arg Pro Val Pro Val Arg Asn His His His 20 2540828PRTartificialSynthetic polypeptide 408Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Gly Met Ser Arg Leu Arg Ser Trp His His His 20 2540928PRTartificialSynthetic polypeptide 409Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Pro Ser Gln Leu Ser Ser Arg His His His 20 2541028PRTartificialSynthetic polypeptide 410Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Phe Ser Leu Ser Arg Thr Ser Ser Lys His His His 20 2541128PRTartificialSynthetic polypeptide 411Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15His Arg Val Gln Gln Phe Ser Pro Ala His His His 20 2541228PRTartificialSynthetic polypeptide 412Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Asp Ser Met Leu Thr Phe Arg Arg Ser His His His 20 2541328PRTartificialSynthetic polypeptide 413Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ser Leu Thr Ser Pro Leu Arg Met His His His 20 2541428PRTartificialSynthetic polypeptide 414Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Ala Ser Phe Leu Arg Pro Ile His His His 20 2541528PRTartificialSynthetic polypeptide 415Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Met1 5 10 15Thr Phe Gln Ser Asn Ser Pro Arg Gly His His His 20 2541628PRTartificialSynthetic polypeptide 416Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Pro Met Thr Leu Arg Gln Pro Val His His His 20 2541728PRTartificialSynthetic polypeptide 417Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Arg Pro Met Ser Arg Val Ile Met Ser His His His 20 2541828PRTartificialSynthetic polypeptide 418Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Tyr Gly Phe Ser Arg Pro Phe Ser Lys His His His 20 2541928PRTartificialSynthetic polypeptide 419Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Cys Phe Ala Phe Met Leu Pro His His His 20 2542028PRTartificialSynthetic polypeptide 420Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Phe Ser Gly Ala Phe Arg Gln Ser Gln His His His 20 2542128PRTartificialSynthetic polypeptide 421Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Arg Ala Gly Ser Phe Ser Ala Ala Pro His His His 20 2542228PRTartificialSynthetic polypeptide 422Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15His Ser Met Ala Pro Pro Ser Arg Arg His His His 20 2542328PRTartificialSynthetic polypeptide 423Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ser Gly Thr Phe Gly Asn Ile Gly His His His 20 2542428PRTartificialSynthetic polypeptide 424Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Met Ala Ser Thr Pro Leu Ala His His His 20 2542528PRTartificialSynthetic polypeptide 425Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Tyr Pro Leu Ala Pro Arg Leu Arg Asp His His His 20 2542628PRTartificialSynthetic polypeptide 426Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Leu Pro Trp Arg Arg Thr Pro Phe Gln His His His 20 2542728PRTartificialSynthetic polypeptide 427Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Met1 5 10 15Arg Thr Pro Pro Leu Ser Gln Arg Ile His His His 20 2542828PRTartificialSynthetic polypeptide 428Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Ser Ser Tyr Asn Ala Val His His His 20 2542928PRTartificialSynthetic polypeptide 429Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15His Ala Leu Ala Arg Lys Ser Gln Phe His His His 20 2543028PRTartificialSynthetic polypeptide 430Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Phe Ser Ser Pro Ser Ile Thr His His His 20 2543128PRTartificialSynthetic polypeptide 431Val

Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ala Leu Ser Lys Pro Leu Pro Pro His His His 20 2543228PRTartificialSynthetic polypeptide 432Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Pro Ser Ala Pro Lys Met Leu Leu His His His 20 2543328PRTartificialSynthetic polypeptide 433Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Ser Tyr Phe Gln Pro Leu His His His 20 2543428PRTartificialSynthetic polypeptide 434Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Leu Ser Arg Ser Ile Pro His His His His 20 2543528PRTartificialSynthetic polypeptide 435Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Gln Leu His Gln Ser Pro Gly Asn Pro His His His 20 2543628PRTartificialSynthetic polypeptide 436Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ala Ile Ala Arg Pro Pro Tyr Thr His His His 20 2543728PRTartificialSynthetic polypeptide 437Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Ser Thr Val Arg Phe Pro His His His 20 2543828PRTartificialSynthetic polypeptide 438Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ala Phe Ser Ser Pro Leu Ser Asn His His His 20 2543928PRTartificialSynthetic polypeptide 439Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Arg Thr Pro Thr Ile Gln Arg Asp Ser His His His 20 2544028PRTartificialSynthetic polypeptide 440Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ala Val Ser Arg Thr Val Pro Thr His His His 20 2544128PRTartificialSynthetic polypeptide 441Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Gln Ser Met Ala Val Pro Ile Ser Thr His His His 20 2544228PRTartificialSynthetic polypeptide 442Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Gln Pro Ser Arg Gly Phe Met Leu Ile His His His 20 2544328PRTartificialSynthetic polypeptide 443Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Met Val Phe Pro Ala Lys Val His His His 20 2544428PRTartificialSynthetic polypeptide 444Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Thr Leu Lys Gly Pro Glu His His His 20 2544528PRTartificialSynthetic polypeptide 445Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Phe Pro Phe Ser Arg Gln Pro Asn Ala His His His 20 2544628PRTartificialSynthetic polypeptide 446Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ala Leu Thr Ser Ile Ser Gly Met His His His 20 2544728PRTartificialSynthetic polypeptide 447Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Gly Met Ser Leu Asn Val Thr Arg His His His 20 2544828PRTartificialSynthetic polypeptide 448Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15His Trp Arg Thr Gln Arg Pro Pro Glu His His His 20 2544928PRTartificialSynthetic polypeptide 449Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Phe Ser Ser Pro Pro Gly Pro His His His 20 2545028PRTartificialSynthetic polypeptide 450Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ile1 5 10 15Phe Pro Ile Glu Ala Ser Ala Arg Arg His His His 20 2545128PRTartificialSynthetic polypeptide 451Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Ser Met Ala Leu Arg Pro Arg Val His His His 20 2545228PRTartificialSynthetic polypeptide 452Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ala Phe Ser Ser Thr Pro Ala Met His His His 20 2545328PRTartificialSynthetic polypeptide 453Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Val Leu Gln Gly Pro Thr His His His 20 2545428PRTartificialSynthetic polypeptide 454Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Thr Ser Pro Pro Tyr Ile His His His 20 2545528PRTartificialSynthetic polypeptide 455Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Asn Arg Pro Gln Ser Thr Lys Asn Ile His His His 20 2545628PRTartificialSynthetic polypeptide 456Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ala Leu Thr Met Thr Pro Ser Phe His His His 20 2545728PRTartificialSynthetic polypeptide 457Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Thr Arg Leu Phe Ala Phe Met Leu Thr His His His 20 2545828PRTartificialSynthetic polypeptide 458Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ala Met Ser Pro Ile Pro Arg Gln His His His 20 2545928PRTartificialSynthetic polypeptide 459Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Met Gly Ser Met Trp Gln Leu His His His 20 2546028PRTartificialSynthetic polypeptide 460Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Ser Met Thr Arg Ser Ser Pro Leu His His His 20 2546128PRTartificialSynthetic polypeptide 461Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Ser Phe Thr Arg Gln Pro Leu Pro His His His 20 2546228PRTartificialSynthetic polypeptide 462Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Arg Val Pro Ser Pro Ala Ser Gln Thr His His His 20 2546328PRTartificialSynthetic polypeptide 463Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Ser Phe Ser Lys Pro Arg Phe Ser His His His 20 2546428PRTartificialSynthetic polypeptide 464Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Thr Gln Phe Ser Ser Val His His His 20 2546528PRTartificialSynthetic polypeptide 465Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Cys Phe Ser Ser Pro Val Ala Leu His His His 20 2546628PRTartificialSynthetic polypeptide 466Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Ala Ser Ser Trp Trp Leu Phe Pro Ser His His His 20 2546728PRTartificialSynthetic polypeptide 467Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Pro Pro Gln Gln Gln Ala Leu Leu Ser His His His 20 2546828PRTartificialSynthetic polypeptide 468Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Gly Phe Ser Met Ala Phe Phe Pro His His His 20 2546928PRTartificialSynthetic polypeptide 469Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Leu Ala Met Ser Arg Pro Gln Ala Ser His His His 20 2547028PRTartificialSynthetic polypeptide 470Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Tyr Ala Leu Thr Thr Phe Gln Ser Val His His His 20 2547128PRTartificialSynthetic polypeptide 471Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15His Ala Phe Thr Arg Pro Phe Arg Val His His His 20 2547228PRTartificialSynthetic polypeptide 472Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ala Phe Ser Ser Pro Ser Gly Ser His His His 20 2547328PRTartificialSynthetic polypeptide 473Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Ala Leu Ala Arg Ser Pro Arg Val His His His 20 2547428PRTartificialSynthetic polypeptide 474Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ala Met Ser Ser Pro Phe Arg Pro His His His 20 2547528PRTartificialSynthetic polypeptide 475Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Thr Phe Ala Arg Ser Phe Met Leu Thr His His His 20 2547628PRTartificialSynthetic polypeptide 476Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Pro Leu Ser Ser Arg Ala Phe Met Leu His His His 20 2547728PRTartificialSynthetic polypeptide 477Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Ser Thr Ser Pro Ile Leu His His His 20 2547828PRTartificialSynthetic polypeptide 478Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Gly Leu Gln Leu Pro Gln Pro Phe His His His 20 2547928PRTartificialSynthetic polypeptide 479Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Met Ser Leu Ser Ser Asp Leu His His His 20 2548028PRTartificialSynthetic polypeptide 480Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Phe Pro Leu Ala Arg Arg Pro Ile Asn His His His 20 2548128PRTartificialSynthetic polypeptide 481Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Cys Arg Ala Met Thr Leu Pro Arg His His His 20 2548228PRTartificialSynthetic polypeptide 482Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Tyr Pro Phe Ser Arg Ala Gly Pro Pro His His His 20 2548328PRTartificialSynthetic polypeptide 483Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Asn Gln Gln Ala Leu Pro Phe Gln Leu His His His 20 2548428PRTartificialSynthetic polypeptide 484Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Trp Ser Met Ser Leu Arg Ser His Ser His His His 20 2548528PRTartificialSynthetic polypeptide 485Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Pro Gln Val Val Thr Arg Lys Asp Leu His His His 20 2548628PRTartificialSynthetic polypeptide 486Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Arg Asn Ala His Ala Met Ala Ser Ala His His His 20 2548728PRTartificialSynthetic polypeptide 487Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Gly Ser Phe Asn Val Thr Pro His His His 20 2548828PRTartificialSynthetic polypeptide 488Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Pro Leu Ser Arg Val Pro Val Phe His His His 20 2548928PRTartificialSynthetic polypeptide 489Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Lys Arg Met Pro Pro Pro Ile Ser Gln His His His 20 2549028PRTartificialSynthetic polypeptide 490Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ser Met Ser Ser Leu Pro Ser Pro His His His 20 2549128PRTartificialSynthetic polypeptide 491Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ser Ser Ser Ser Ile Phe Pro Leu His His His 20 2549228PRTartificialSynthetic polypeptide 492Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Arg1 5 10 15Ser Ala His Ala Met Ser Ile Gln Thr His His His 20 2549328PRTartificialSynthetic polypeptide 493Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Tyr Cys Phe Ser Ala Arg Ile Ile Arg His His His 20 2549428PRTartificialSynthetic polypeptide 494Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15His Leu Ser Pro Leu Gln Pro Gln Gln His His His 20 2549528PRTartificialSynthetic polypeptide 495Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Ser Phe Ser Arg Phe Pro Gly Leu His His His 20 2549628PRTartificialSynthetic polypeptide 496Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Ser Met Ser Leu Arg Pro Gln Phe His His His 20 2549728PRTartificialSynthetic polypeptide 497Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Pro Arg Ala Arg Pro Val Pro Pro His His His 20 2549828PRTartificialSynthetic polypeptide 498Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Ser Ala Leu Ser Pro Tyr His His His 20 2549928PRTartificialSynthetic polypeptide 499Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Val Arg Gln Leu His Thr Asn Leu Arg His His His 20 2550028PRTartificialSynthetic polypeptide 500Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Thr Thr Ser Thr Pro Tyr Gln Ser Pro His His His 20 2550128PRTartificialSynthetic polypeptide 501Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Asn Ala Leu Thr Phe Leu Pro Ser Gln His His His 20 2550228PRTartificialSynthetic polypeptide 502Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Arg Ser Leu Ser Ser Pro Leu Thr Leu His His His

20 2550328PRTartificialSynthetic polypeptide 503Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Pro Pro Thr Val Gly Leu Arg Gln His His His 20 2550428PRTartificialSynthetic polypeptide 504Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Ala Leu Ser Pro Met Ser Trp Gln His His His 20 2550528PRTartificialSynthetic polypeptide 505Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Phe Pro Phe Ser Arg Pro Leu Leu Arg His His His 20 2550628PRTartificialSynthetic polypeptide 506Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Pro Arg Cys Leu Ser Met Ser Leu Gly His His His 20 2550728PRTartificialSynthetic polypeptide 507Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Gln Pro Ser Phe His Pro Ile Ser Arg His His His 20 2550828PRTartificialSynthetic polypeptide 508Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Lys Ala Phe Ser Ser Phe Gln Ala Ser His His His 20 2550928PRTartificialSynthetic polypeptide 509Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Tyr Ser Met Ser Gln Ser Gly Leu Thr His His His 20 2551028PRTartificialSynthetic polypeptide 510Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Gln Ala Leu Thr Thr Arg Gly Leu Ala His His His 20 2551128PRTartificialSynthetic polypeptide 511Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Lys Ser Leu Thr Arg Pro Ala Phe Leu His His His 20 2551228PRTartificialSynthetic polypeptide 512Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Gln Ser Arg Leu Arg Val Tyr Pro Pro His His His 20 2551328PRTartificialSynthetic polypeptide 513Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Ala Ile Gly Phe Met Leu Leu Arg Tyr His His His 20 2551428PRTartificialSynthetic polypeptide 514Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Gly Thr Leu Val Arg Pro Arg Pro His His His 20 2551528PRTartificialSynthetic polypeptide 515Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ile1 5 10 15Arg Arg Pro Val Asp Pro Val Met Pro His His His 20 2551628PRTartificialSynthetic polypeptide 516Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Pro Leu Arg Gln Thr His Arg Tyr Pro His His His 20 2551728PRTartificialSynthetic polypeptide 517Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15His Ser Met Gln Arg Pro Thr Gly Arg His His His 20 2551828PRTartificialSynthetic polypeptide 518Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Arg1 5 10 15His Thr Gln Leu Ser Ser Ser Thr Ser His His His 20 2551928PRTartificialSynthetic polypeptide 519Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Cys Gly Phe Ser Arg Leu Ser Lys Ala His His His 20 2552028PRTartificialSynthetic polypeptide 520Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Ser Phe Ser Gln Leu Pro His Ile His His His 20 2552128PRTartificialSynthetic polypeptide 521Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Ser Met Ser Gln Leu Arg Pro Phe His His His 20 2552228PRTartificialSynthetic polypeptide 522Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Thr Thr Phe Ala Leu Gln Ser Ser His His His 20 2552328PRTartificialSynthetic polypeptide 523Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Gln Ser Met Ser Ile Arg His Asn Asn His His His 20 2552428PRTartificialSynthetic polypeptide 524Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Ser Arg Phe Arg Thr Thr Pro Pro Ser His His His 20 2552528PRTartificialSynthetic polypeptide 525Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Val Ser Met Ser Arg Tyr Gln Leu Ser His His His 20 2552628PRTartificialSynthetic polypeptide 526Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Gly Ala Ser Arg Leu Arg Ile Leu His His His 20 2552728PRTartificialSynthetic polypeptide 527Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Trp Ser Leu Ser Arg Pro Arg Leu Leu His His His 20 2552828PRTartificialSynthetic polypeptide 528Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Arg Ser Thr Lys Leu Thr Pro Ser His His His 20 2552928PRTartificialSynthetic polypeptide 529Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Val Ser Val Ala Phe Met Leu Met His His His 20 2553028PRTartificialSynthetic polypeptide 530Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Leu Gly Arg Ser Met Ala Pro Gly Pro His His His 20 2553128PRTartificialSynthetic polypeptide 531Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Val His Arg Arg Asp Ser Ser Ser Leu His His His 20 2553228PRTartificialSynthetic polypeptide 532Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Leu Gly Phe Ser Arg Leu Thr Ser Leu His His His 20 2553328PRTartificialSynthetic polypeptide 533Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Ala Leu Ser Arg Arg Val Pro Gln His His His 20 2553428PRTartificialSynthetic polypeptide 534Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Tyr Pro Ala Ser Trp Pro Arg Leu Arg His His His 20 2553528PRTartificialSynthetic polypeptide 535Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Val Ser Leu Ala Val Thr Pro Ser His His His 20 2553628PRTartificialSynthetic polypeptide 536Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Asn Pro Phe Ser Ser Leu Ser Gln Gln His His His 20 2553728PRTartificialSynthetic polypeptide 537Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Arg1 5 10 15Pro Leu Pro Arg Pro Phe Ala Gly Asn His His His 20 2553828PRTartificialSynthetic polypeptide 538Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Phe Ser Met Thr Gln Tyr Leu Pro Gln His His His 20 2553928PRTartificialSynthetic polypeptide 539Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Ala Leu Ser Arg Ser Phe Tyr Pro His His His 20 2554028PRTartificialSynthetic polypeptide 540Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Gln Gln Arg Cys Phe Ala Met His Ile His His His 20 2554128PRTartificialSynthetic polypeptide 541Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ile1 5 10 15Lys His Phe Tyr Asn Ser Arg Pro Ser His His His 20 2554228PRTartificialSynthetic polypeptide 542Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Thr Arg Leu Pro Lys Glu Ser Ser Pro His His His 20 2554328PRTartificialSynthetic polypeptide 543Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Pro Ala Gln Pro Arg Val Thr Arg Thr His His His 20 2554428PRTartificialSynthetic polypeptide 544Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Arg Ser Met Thr Leu Asn Thr Ser Thr His His His 20 2554528PRTartificialSynthetic polypeptide 545Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Asp Thr Phe Ser Tyr Ser Ser Gln Asp His His His 20 2554628PRTartificialSynthetic polypeptide 546Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Arg Asn Pro Gln Leu Pro Ser Ser Ala His His His 20 2554728PRTartificialSynthetic polypeptide 547Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Arg Pro Asp Arg Thr Pro Pro Ser Ser His His His 20 2554828PRTartificialSynthetic polypeptide 548Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Ser His Thr Ile Leu Pro Leu Pro Ala His His His 20 2554928PRTartificialSynthetic polypeptide 549Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Ala Phe Gln Pro Met Val Ser Ser His His His 20 2555028PRTartificialSynthetic polypeptide 550Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Ser Arg Arg Leu Pro Ile Leu Pro Leu His His His 20 2555128PRTartificialSynthetic polypeptide 551Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Gln Ala Tyr Leu Pro Ala Pro Gln Leu His His His 20 2555228PRTartificialSynthetic polypeptide 552Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Arg Pro Arg Glu Thr Leu Phe Leu His His His 20 2555328PRTartificialSynthetic polypeptide 553Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ala Ala Ser Val Val Arg Ser Arg Asp His His His 20 2555428PRTartificialSynthetic polypeptide 554Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Arg Gly Ala Ala Pro Lys Phe Ser Val His His His 20 2555528PRTartificialSynthetic polypeptide 555Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Phe1 5 10 15Arg His Gln Pro Ala Ser Val Ser Thr His His His 20 2555628PRTartificialSynthetic polypeptide 556Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Thr Asn Ala Ile Ala Phe Phe Leu Gln His His His 20 2555728PRTartificialSynthetic polypeptide 557Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Lys Ser Leu Arg Ser Asp Thr Pro Asn His His His 20 2555828PRTartificialSynthetic polypeptide 558Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ile1 5 10 15Lys Arg Pro Leu Pro Leu Ala Pro Thr His His His 20 2555928PRTartificialSynthetic polypeptide 559Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Ser Ser Lys Ser Arg Phe Met Leu His His His 20 2556028PRTartificialSynthetic polypeptide 560Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Trp Lys Pro Arg Leu Leu Pro Pro Gln His His His 20 2556128PRTartificialSynthetic polypeptide 561Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Arg Gly Phe Met Leu Thr Leu Arg Tyr His His His 20 2556228PRTartificialSynthetic polypeptide 562Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Lys Ala Arg Gly Ile Met Pro Val Phe His His His 20 2556328PRTartificialSynthetic polypeptide 563Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Leu Pro Arg Leu Thr Ser Gln Ser His His His 20 2556428PRTartificialSynthetic polypeptide 564Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Ser Ser Ala Phe Ser Tyr Met Leu Ser His His His 20 2556528PRTartificialSynthetic polypeptide 565Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Phe Ser Ser Gln Arg Phe Leu Arg Pro His His His 20 2556628PRTartificialSynthetic polypeptide 566Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Ser Ser Asn Thr Ser Arg Arg Phe Pro His His His 20 2556728PRTartificialSynthetic polypeptide 567Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Gln Thr Ala Ala Thr Ala Pro Pro Arg His His His 20 2556828PRTartificialSynthetic polypeptide 568Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gly1 5 10 15Ala Pro Leu Ser Trp Arg Arg Ser Tyr His His His 20 2556928PRTartificialSynthetic polypeptide 569Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Arg Ser Val Trp Cys Ile Pro Arg Pro His His His 20 2557028PRTartificialSynthetic polypeptide 570Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Lys Ala Cys Leu Arg Pro Leu Gln Thr His His His 20 2557128PRTartificialSynthetic polypeptide 571Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Leu Ala Ser Ser His Arg His Arg Pro His His His 20 2557228PRTartificialSynthetic polypeptide 572Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Arg Ala Asp Ser Leu Ala Pro Lys Ser His His His 20 2557328PRTartificialSynthetic polypeptide 573Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Val Pro Gln Phe Ser Gly Arg Ser Arg His His His 20 2557428PRTartificialSynthetic polypeptide 574Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10

15Tyr Pro Ala Arg Phe Pro Ala Lys Thr His His His 20 2557528PRTartificialSynthetic polypeptide 575Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asn1 5 10 15Phe Met Leu Arg His Pro Gln Thr Phe His His His 20 2557628PRTartificialSynthetic polypeptide 576Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Tyr1 5 10 15Val Pro Arg Phe Pro Pro Lys Ser Ala His His His 20 2557728PRTartificialSynthetic polypeptide 577Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Ser Pro Met Ser Arg Thr Arg Tyr Val His His His 20 2557828PRTartificialSynthetic polypeptide 578Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Tyr Pro Leu Thr Lys Pro Tyr Arg Pro His His His 20 2557928PRTartificialSynthetic polypeptide 579Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ser Tyr Trp Ser His Arg Lys Pro Pro His His His 20 2558028PRTartificialSynthetic polypeptide 580Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Pro Arg Thr Phe Ala Phe Phe Leu Met His His His 20 2558128PRTartificialSynthetic polypeptide 581Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Leu1 5 10 15Gly Pro Gly Ile Arg Lys Lys Pro Ala His His His 20 2558228PRTartificialSynthetic polypeptide 582Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Arg Leu Cys Val Ala Lys Val Ala Gly His His His 20 2558328PRTartificialSynthetic polypeptide 583Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Arg1 5 10 15Ser Leu Pro Ala Ser Gly Ala Ser Arg His His His 20 2558428PRTartificialSynthetic polypeptide 584Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ala1 5 10 15Ser Pro Arg Val Lys Ser Tyr Ser Pro His His His 20 2558528PRTartificialSynthetic polypeptide 585Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Ser Arg Thr Phe Ala Phe Tyr Leu Val His His His 20 2558628PRTartificialSynthetic polypeptide 586Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Gln1 5 10 15Gln Glu Phe Ala Met Ala His His His His His His 20 2558728PRTartificialSynthetic polypeptide 587Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Pro1 5 10 15Gln Ser Ser Lys Ala Phe Phe Leu Asn His His His 20 2558828PRTartificialSynthetic polypeptide 588Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Val1 5 10 15Lys Ala Leu Arg Gly Ser Tyr Pro Thr His His His 20 2558928PRTartificialSynthetic polypeptide 589Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Thr1 5 10 15Gln Pro Ser Gln Val Arg Tyr Met Leu His His His 20 2559028PRTartificialSynthetic polypeptide 590Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Ala Arg Gly Gln His Val Arg Pro Pro His His His 20 2559128PRTartificialSynthetic polypeptide 591Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15Thr Arg Cys Pro Gly Phe Phe Leu Gln His His His 20 2559228PRTartificialSynthetic polypeptide 592Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Cys1 5 10 15Pro Ser Val Phe Ser Arg Thr Pro Pro His His His 20 2559328PRTartificialSynthetic polypeptide 593Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Asp1 5 10 15Ala Ser Ser Trp Arg His Phe Leu Ser His His His 20 2559418PRTartificial sequencethe wild-type M13 signal peptide N-terminal to gene III 594Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser1 5 10 15His Ser59522PRTartificial sequencethe pelB signal sequence of Pectobacterium wasabiae 595Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala 2059630PRTartificial sequenceSignal peptide 596Val Lys Lys Leu Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala1 5 10 15Ala Gln Pro Ala Met Ala His His His His His His Gly His 20 25 3059730PRTartificial sequenceSignal peptide 597Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Met Ala His His His His His His Gly His 20 25 3059830PRTartificial sequenceSignal peptide 598Val Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His His His Gly His 20 25 30

Claims

1. A nucleic acid library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of expression constructs, each of which includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of: (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.0X.sub- .10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

2. The nucleic acid library of claim 1, wherein each of the expression constructs further includes a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence.

3. The nucleic acid library of claim 2, wherein the expression construct is a phagemid.

4. The nucleic acid library of claim 1, wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond.

5. The nucleic acid library of claim 4, wherein the first variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

6. The nucleic acid library of claim 5, wherein the second variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

7. A host cell library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of host cells each containing an expression construct that includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of: (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub- .10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

8. The host cell library of claim 6, wherein each of the expression constructs further includes a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence.

9. A phage library for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, the library comprising a plurality of phage particles each containing a disulfide-stabilized single chain antibody fused with a coat protein on the surface of said phage, wherein the phage library is prepared by the steps of: providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the plurality of phage particles, wherein the expression construct that includes a first nucleotide sequence encoding a signal peptide, a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, and a third nucleotide sequence encoding a phage envelop protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence, wherein the signal peptide has the amino acid sequence of (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub- .10AAQPAMAHHHHHHGH (SEQ ID NO:1), (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:2), or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:3), each of X.sub.1-X.sub.10 in (a), (b), and (c) being one of the 20 naturally occurring amino acid residues.

10. An isolated nucleic acid, comprising a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, wherein the signal peptide has the amino acid sequence of: (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X- .sub.10AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:597), in which X.sub.1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:598), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

11. The nucleic acid of claim 10, further comprising a third nucleotide encoding a phage coat protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence.

12. The nucleic acid of claim 10, wherein the nucleic acid is an expression vector for expression a fusion protein containing the signal peptide and the single chain antibody.

13. The nucleic acid of claim 10, wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond.

14. The nucleic acid library of claim 13, wherein the first variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

15. The nucleic acid library of claim 13, wherein the second variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

16. The nucleic acid of claim 12, wherein the expression vector is a phagemid.

17. A host cell containing the nucleic acid of claim 13.

18. A phage containing a disulfide-stabilized single chain antibody fused with its coat protein on the surface, wherein the phage is prepared by the method comprising the steps of: providing a host cell of claim 17, and culturing the host cell in a medium under conditions allowing expression of the phage.

19. A method for producing a disulfide-stabilized single chain antibody, comprising providing a host cell containing an expression construct, and culturing the host cell in a medium under conditions allowing expression of the disulfide-stabilized single chain antibody, wherein the expression construct includes a first nucleotide sequence encoding a signal peptide, and a second nucleotide sequence encoding a single chain antibody capable of forming an interface disulfide bond, the second nucleotide sequence being located 3' downstream to the first nucleotide, and wherein the signal peptide has the amino acid sequence of (a) VKKLLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub- .10AAQPAMAHHHHHHGH (SEQ ID NO:596), in which X.sub.1 is A, C, F, G, I, L, M, P, Q, S, V, W, or Y; X.sub.2 is A, D, F, G, H, I, L, M, N, P, S, T, V, or W; X.sub.3 is A, F, G, L, M, P, Q, R, S, T, V, or W; X.sub.4 is A, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.8 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.9 is A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; (b) VKKLLFAIPLX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9- X.sub.10MAHHHHHHGH (SEQ ID NO:597), in which X.sub.1 A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; X.sub.2 is A, C, D, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, D, F, G, H, I, L, M, N, P, O, R, S, T, V, W, or Y; X.sub.4 is A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.6 is A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, C, F, G, I, K, L, M, N, P, Q, R, S, T, or V; X.sub.9 is A, C, D, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, C, D, E, F, G, H, L, M, P, Q, R, S, or T; or (c) VKKLLFAIPLVVPFYX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.- sub.9X.sub.10HHHGH (SEQ ID NO:598), in which X.sub.1 is A, C, D, F, G, I, L, M, N, P, Q, R, S, T, V, or Y; X.sub.2 is A, C, D, F, G, H, K, L, N, P, Q, R, S, T, V, W, or Y; X.sub.3 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.4 is A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.5 is A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; X.sub.6 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; X.sub.7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; X.sub.8 is A, E, F, G, H, I, K, L, M, N, P, O, R, S, T, V, W, or Y; X.sub.9 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and X.sub.10 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y.

20. The method of claim 19, wherein the single chain antibody contains a first variable region, a second variable region, and a protein linker connecting the first and the second variable region, wherein the first and the second variable region are stabilized by an interface disulfide bond.

21. The method of claim 20, wherein the first variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

22. The method of claim 20, wherein the second variable region is a heavy chain variable region (V.sub.H) or a light chain variable region (V.sub.L).

23. The method of claim 19, further comprising, after the culturing step, collecting the medium for isolating the disulfide-stabilized single chain antibody.

24. The method of claim 19, wherein the expression construct is a phagemid that further includes a third nucleotide encoding a phage envelope protein, the third nucleotide sequence being located 3' downstream to the second nucleotide sequence.

25. The method of claim 24, further comprising, after the culturing step, collecting the medium for isolating phage particles that display the disulfide-stabilized single chain antibody.

26. An array of disulfide-stabilized single chain antibodies produced by the method of claim 19 coated on a solid surface.

27. The array of claim 26, wherein the solid surface is selected from the group consisting of silicon, plastic, nylon, glass, ceramic, photoresist, and rubber surface.

28. An isolated signal peptide that facilitates production of a disulfide-stabilized single chain antibody, which is one selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-593.

29. The signal peptide of claim 28, which is one selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 381-429 and 476.

30. An isolated nucleic acid for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, or for facilitating production of a disulfide-stabilized single chain antibody, which is a nucleic acid encoding a signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-593.

31. The nucleic acid of claim 30, which is a nucleic acid encoding a signal peptide selected from the group consisting of the peptides having the amino acid sequences set forth in SEQ ID NOS: 5-16, 18-19, 21-29, 31-36, 38-42, 45, 48-53, 55, 57-64, 255-304, 381-429 and 476.