U.S. patent application number 13/565559 was filed with the patent office on 2013-10-10 for artificial antibody polypeptides.
The applicant listed for this patent is Shohei Koide. Invention is credited to Shohei Koide.
Application Number | 20130267676 13/565559 |
Document ID | / |
Family ID | 22811231 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130267676 |
Kind Code |
A1 |
Koide; Shohei |
October 10, 2013 |
ARTIFICIAL ANTIBODY POLYPEPTIDES
Abstract
The present invention provides a fibronectin type III (Fn3)
molecule, wherein the Fn3 contains a stabilizing mutation. The
present invention also provides Fn3 polypeptide monobodies, nucleic
acid molecules encoding monobodies, and variegated nucleic acid
libraries encoding such monobodies. Also provided are methods of
preparing a Fn3 polypeptide monobody, and kits to perform the
methods.
Inventors: |
Koide; Shohei; (Rochester,
NY) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Koide; Shohei |
Rochester |
NY |
US |
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|
Family ID: |
22811231 |
Appl. No.: |
13/565559 |
Filed: |
August 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09903412 |
Jul 11, 2001 |
8263741 |
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13565559 |
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60217474 |
Jul 11, 2000 |
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Current U.S.
Class: |
530/300 ;
435/183; 435/252.33; 435/320.1; 435/69.1; 506/1; 506/17; 506/18;
506/2; 506/6; 530/350; 536/23.5 |
Current CPC
Class: |
C40B 30/04 20130101;
C12N 15/1044 20130101; C07K 2317/565 20130101; C07K 14/78 20130101;
C07K 16/00 20130101; G01N 33/6845 20130101; C12N 15/1037
20130101 |
Class at
Publication: |
530/300 ;
530/350; 536/23.5; 435/320.1; 435/183; 435/252.33; 435/69.1;
506/17; 506/18; 506/2; 506/6; 506/1 |
International
Class: |
C07K 14/78 20060101
C07K014/78; C12N 15/10 20060101 C12N015/10 |
Goverment Interests
[0001] Portions of the present invention were made with support of
the United States Government via a grant from the National
Institutes of Health under grant number GM 55042 The U.S.
Government therefore may have certain rights in the invention.
Claims
1. A fibronectin type III (Fn3) molecule, wherein the Fn3 comprises
a stabilizing mutation as compared to a wild-type Fn3.
2. The Fn3 of claim 1, wherein the stabilizing mutation comprises
at least one aspartic acid (Asp) residue that has been deleted or
substituted with at least one other amino acid residue.
3. The Fn3 of claim 2, wherein Asp 7 or Asp 23, or both, have been
deleted or substituted with at least one other amino acid
residue.
4. The Fn3 of claim 3, wherein Asp 7 or Asp 23, or both, have been
substituted with an asparagine (Asn) or lysine (Lys) residue.
5. The Fn3 of claim 1, wherein the stabilizing mutation comprises
at least one glutamic acid (Glu) residue that has been deleted or
substituted with at least one other amino acid residue.
6. The Fn3 of claim 5, wherein Glu 9 has been deleted or
substituted with at least one other amino acid residue.
7. The Fn3 of claim 6, wherein Glu 9 has been substituted with an
asparagine (Asn) or lysine (Lys) residue.
8. The Fn3 of claim 2, wherein Asp 7, Asp 23, and Glu 9 have been
deleted or substituted with at least one other amino acid
residue.
9. A fibronectin type III (Fn3) polypeptide monobody comprising a
plurality of Fn3 .beta.-strand domain sequences that are linked to
a plurality of loop region sequences, wherein one or more of the
monobody loop region sequences vary by deletion, insertion or
replacement of at least two amino acids from the corresponding loop
region sequences in wild-type Fn3; wherein the .beta.-strand
domains of the monobody have at least a 50% total amino acid
sequence homology to the corresponding amino acid sequence of
wild-type Fn3's .beta.-strand domain sequences; and wherein the Fn3
comprises a stabilizing mutation.
10. An isolated nucleic acid molecule encoding the Fn3 molecule of
claim 9.
11. An expression vector comprising an expression cassette operably
linked to the nucleic acid molecule of claim 10.
12. A host cell comprising the vector of claim 11.
13. The monobody of claim 9, wherein at least one loop region is
capable of binding to a specific binding partner (SBP) to form a
polypeptide:SBP complex having a dissociation constant of less than
10.sup.-6 moles/liter.
14. The monobody of claim 9, wherein at least one loop region is
capable of catalyzing a chemical reaction with a catalyzed rate
constant (k.sub.cat) and an uncatalyzed rate constant (k.sub.uncat)
such that the ratio of k.sub.cat/k.sub.uncat is greater than
10.
15. The monobody of claim 9, wherein one or more of the loop
regions comprise amino acid residues: i) from 15 to 16 inclusive in
an AB loop; ii) from 22 to 30 inclusive in a BC loop; iii) from 39
to 45 inclusive in a CD loop; iv) from 51 to 55 inclusive in a DE
loop; v) from 60 to 66 inclusive in an EF loop; and vi) from 76 to
87 inclusive in an FG loop.
16. The monobody of claim 9, wherein the monobody loop region
sequences vary from the wild-type Fn3 loop region sequences by the
deletion or replacement of at least 2 amino acids.
17. The monobody of claim 9, wherein the monobody loop region
sequences vary from the wild-type Fn3 loop region sequences by the
insertion of from 3 to 25 amino acids.
18. An isolated nucleic acid molecule encoding the polypeptide
monobody of claim 1.
19. An expression vector comprising an expression cassette operably
linked to the nucleic acid molecule of claim 18.
20. The expression vector of claim 19, wherein the expression
vector is an M13 phage-based plasmid.
21. A host cell comprising the vector of claim 19.
22. A method of preparing a fibronectin type III (Fn3) polypeptide
monobody comprising the steps of: a) providing a DNA sequence
encoding a plurality of Fn3 .beta.-strand domain sequences that are
linked to a plurality of loop region sequences, wherein at least
one loop region contains a unique restriction enzyme site, and
wherein at least one of the plurality of Fn3 .beta.-strand domain
sequences are more stable at neutral pH than wild-type Fn3; b)
cleaving the DNA sequence at the unique restriction site; c)
inserting into the restriction site a DNA segment known to encode a
peptide capable of binding to a specific binding partner (SBP) or a
transition state analog compound (TSAC) so as to yield a DNA
molecule comprising the insertion and the DNA sequence of (a); and
d) expressing the DNA molecule so as to yield polypeptide
monobody.
23. A method of preparing a fibronectin type III (Fn3) polypeptide
monobody comprising the steps of: (a) providing a replicatable DNA
sequence encoding a plurality of Fn3 .beta.-strand domain sequences
that are linked to a plurality of loop region sequences, wherein
the nucleotide sequence of at least one loop region is known, and
wherein at least one of the plurality of Fn3 .beta.-strand domain
sequences are more stable at neutral pH than wild-type Fn3; (b)
preparing polymerase chain reaction (PCR) primers sufficiently
complementary to the known loop sequence so as to be hybridizable
under PCR conditions, wherein at least one of the primers contains
a modified nucleic acid sequence to be inserted into the DNA; (c)
performing polymerase chain reaction using the DNA sequence of (a)
and the primers of (b); (d) annealing and extending the reaction
products of (c) so as to yield a DNA product; and (e) expressing
the polypeptide monobody encoded by the DNA product of (d).
24. A method of preparing a fibronectin type III (Fn3) polypeptide
monobody comprising the steps of: a) providing a replicatable DNA
sequence encoding a plurality of Fn3 .beta.-strand domain sequences
that are linked to a plurality of loop region sequences, wherein
the nucleotide sequence of at least one loop region is known, and
wherein at least one of the plurality of Fn3 .beta.-strand domain
sequences are more stable at neutral pH than wild-type Fn3; b)
performing site-directed mutagenesis of at least one loop region so
as to create a DNA sequence comprising an insertion mutation; and
c) expressing the polypeptide monobody encoded by the DNA sequence
comprising the insertion mutation.
25. A kit for performing the method of any one of claims 22-24,
comprising a replicatable DNA encoding a plurality of Fn3
.beta.-strand domain sequences that are linked to a plurality of
loop region sequences, wherein at least one of the plurality of Fn3
.beta.-strand domain sequences are more stable at neutral pH than
wild-type Fn3.
26. A variegated nucleic acid library encoding Fn3 polypeptide
monobodies comprising a plurality of nucleic acid species each
comprising a plurality of loop regions, wherein the species encode
a plurality of Fn3 .beta.-strand domain sequences that are linked
to a plurality of loop region sequences, wherein one or more of the
loop region sequences vary by deletion, insertion or replacement of
at least two amino acids from corresponding loop region sequences
in wild-type Fn3; wherein the .beta.-strand domain sequences of the
monobody have at least a 50% total amino acid sequence homology to
the corresponding amino acid sequences of .beta.-strand domain
sequences of the wild-type Fn3; and wherein the Fn3 is more stable
at neutral pH than wild-type Fn
27. The variegated nucleic acid library of claim 26, wherein one or
more of the loop regions encodes: i) an AB amino acid loop from
residue 15 to 16 inclusive; ii) a BC amino acid loop from residue
22 to 30 inclusive; iii) a CD amino acid loop from residue 39 to 45
inclusive; iv) a DE amino acid loop from residue 51 to 55
inclusive; v) an EF amino acid loop from residue 60 to 66
inclusive; and vi) an FG amino acid loop from residue 76 to 87
inclusive.
28. The variegated nucleic acid library of claim 26, wherein the
loop region sequences vary from the wild-type Fn3 loop region
sequences by the deletion or replacement of at least 2 amino
acids.
29. The variegated nucleic acid library of claim 26, wherein the
monobody loop region sequences vary from the wild-type Fn3 loop
region sequences by the insertion of from 3 to 25 amino acids.
30. The variegated nucleic acid library of claim 26, wherein a
variegated nucleic acid sequence comprising from 6 to 75 nucleic
acid bases is inserted in any one of the loop regions of the
species.
31. The variegated nucleic acid library of claim 26, wherein the
variegated sequence is constructed so as to avoid one or more
codons selected from the group consisting of those codons encoding
cysteine or the stop codon.
32. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the BC loop.
33. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the DE loop.
34. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the FG loop.
35. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the AB loop.
36. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the CD loop.
37. The variegated nucleic acid library of claim 26, wherein the
variegated nucleic acid sequence is located in the EF loop.
38. A peptide display library derived from the variegated nucleic
acid library of claim 26.
39. A peptide display library of claim 38, wherein the peptide is
displayed on the surface of a bacteriophage or virus.
40. A peptide display library of claim 39, wherein the
bacteriophage is M13 or fd.
41. A method of identifying the amino acid sequence of a
polypeptide molecule capable of binding to a specific binding
partner (SBP) so as to form a polypeptide:SSP complex wherein the
dissociation constant of the polypeptide:SBP complex is less than
10.sup.-6 moles/liter, comprising the steps of: a) providing a
peptide display library according to claim 39; b) contacting the
peptide display library of (a) with an immobilized or separable
SBP; c) separating the peptide:SBP complexes from the free
peptides, d) causing the replication of the separated peptides of
(c) so as to result in a new peptide display library distinguished
from that in (a) by having a lowered diversity and by being
enriched in displayed peptides capable of binding the SBP; e)
optionally repeating steps (b), (c), and (d) with the new library
of (d); and f) determining the nucleic acid sequence of the region
encoding the displayed peptide of a species from (d) and deducing
the peptide sequence capable of binding to the SBP.
42. A method of preparing a variegated nucleic acid library
encoding Fn3 polypeptide monobodies having a plurality of nucleic
acid species each comprising a plurality of loop regions, wherein
the species encode a plurality of Fn3.beta.-strand domain sequences
that are linked to a plurality of loop region sequences, wherein
one or more of the loop region sequences vary by deletion,
insertion or replacement of at least two amino acids from
corresponding loop region sequences in wild-type Fn3, and wherein
the .beta.-strand domain sequences of the monobody have at least a
50% total amino acid sequence homology to the corresponding amino
acid sequences of .beta.-strand domain sequences of the wild-type
Fn3, and wherein the Fn3 comprises a stabilizing mutation
.beta.-strand domain, comprising the steps of a) preparing an Fn3
polypeptide monobody having a predetermined sequence; b) contacting
the polypeptide with a specific binding partner (SBP) so as to form
a polypeptide:SSP complex wherein the dissociation constant of the
polypeptide:SBP complex is less than 10.sup.-6 moles/liter; c)
determining the binding structure of the polypeptide:SBP complex by
nuclear magnetic resonance spectroscopy or X-ray crystallography;
and d) preparing the variegated nucleic acid library, wherein the
variegation is performed at positions in the nucleic acid sequence
which, from the information provided in (c), result in one or more
polypeptides with improved binding to the SBP.
43. A method of identifying the amino acid sequence of a
polypeptide molecule capable of catalyzing a chemical reaction with
a catalyzed rate constant, k.sub.cat, and an uncatalyzed rate
constant, L.sub.uncat, such that the ratio of k.sub.cat/k.sub.uncat
is greater than 10, comprising the steps of: a) providing a peptide
display library according to claim 39; b) contacting the peptide
display library of (a) with an immobilized or separable transition
state analog compound (TSAC) representing the approximate molecular
transition state of the chemical reaction; c) separating the
peptide:TSAC complexes from the free peptides; d) causing the
replication of the separated peptides of (c) so as to result in a
new peptide display library distinguished from that in (a) by
having a lowered diversity and by being enriched in displayed
peptides capable of binding the TSAC; e) optionally repeating steps
(b), (c), and (d) with the new library of (d); and f) determining
the nucleic acid sequence of the region encoding the displayed
peptide of a species from (d) and hence deducing the peptide
sequence.
44. A method of preparing a variegated nucleic acid library
encoding Fn3 polypeptide monobodies having a plurality of nucleic
acid species each comprising a plurality of loop regions, wherein
the species encode a plurality of Fn3 .beta.-strand domain
sequences that are linked to a plurality of loop region sequences,
wherein one or more of the loop region sequences vary by deletion,
insertion or replacement of at least two amino acids from
corresponding loop region sequences in wild-type Fn3, and wherein
the .beta.-strand domain sequences of the monobody have at least a
50% total amino acid sequence homology to the corresponding amino
acid sequences of .beta.-strand domain sequences of the wild-type
Fn3, and wherein the Fn3 comprises a stabilizing mutation
.beta.-strand domain, comprising the steps of a) preparing an Fn3
polypeptide monobody having a predetermined sequence, wherein the
polypeptide is capable of catalyzing a chemical reaction with a
catalyzed rate constant, k.sub.cat, and an uncatalyzed rate
constant, k.sub.uncat, such that the ratio of k.sub.cat/k.sub.uncat
is greater than 10; b) contacting the polypeptide with an
immobilized or separable transition state analog compound (TSAC)
representing the approximate molecular transition state of the
chemical reaction; c) determining the binding structure of the
polypeptide:TSAC complex by nuclear magnetic resonance spectroscopy
or X-ray crystallography; and d) preparing the variegated nucleic
acid library, wherein the variegation is performed at positions in
the nucleic acid sequence which, from the information provided in
(c), result in one or more polypeptides with improved binding to or
stabilization of the TSAC.
45. An isolated polypeptide identified by the method of claim
41.
46. An isolated polypeptide identified by the method of claim
43.
47. A kit for identifying the amino acid sequence of a polypeptide
molecule capable of binding to a specific binding partner (SBP) so
as to form a polypeptide:SSP complex wherein the dissociation
constant of the polypeptide:SBP complex is less than 10.sup.-6
moles/liter, comprising the peptide display library of claim
39.
48. A kit for identifying the amino acid sequence of a polypeptide
molecule capable of catalyzing a chemical reaction with a catalyzed
rate constant, k.sub.cat, and an uncatalyzed rate constant,
k.sub.uncat, such that the ratio of k.sub.cat/k.sub.uncat is
greater than 10, comprising the peptide display library of claim
39.
49. A polypeptide derived by using the kit of claim 47.
50. A polypeptide derived by using the kit of claim 48.
Description
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of the
production and selection of binding and catalytic polypeptides by
the methods of molecular biology. The invention specifically
relates to the generation of both nucleic acid and polypeptide
libraries encoding the molecular scaffolding of a modified
Fibronectin Type III (Fn3) molecule. The invention also relates to
"artificial mini-antibodies" or "monobodies," i.e., polypeptides
containing an Fn3 scaffold onto which loop regions capable of
binding to a variety of different molecular structures (such as
antibody binding sites) have been grafted.
BACKGROUND OF THE INVENTION
Antibody Structure
[0003] A standard antibody (Ab) is a tetrameric structure
consisting of two identical immunoglobulin (Ig) heavy chains and
two identical light chains. The heavy and light chains of an Ab
consist of different domains. Each light chain has one variable
domain (VL) and one constant domain (CL), while each heavy chain
has one variable domain (VH) and three or four constant domains
(CH) (Alzari et al., 1988). Each domain, consisting of .about.110
amino acid residues, is folded into a characteristic
.beta.-sandwich structure formed from two .beta.-sheets packed
against each other, the immunoglobulin fold. The VH and VL domains
each have three complementarity determining regions (CDR1-3) that
are loops, or turns, connecting .beta.-strands at one end of the
domains (FIG. 1: A, C). The variable regions of both the light and
heavy chains generally contribute to antigen specificity, although
the contribution of the individual chains to specificity is not
always equal. Antibody molecules have evolved to bind to a large
number of molecules by using six randomized loops (CDRs). However,
the size of the antibodies and the complexity of six loops
represents a major design hurdle if the end result is to be a
relatively small peptide ligand.
Antibody Substructures
[0004] Functional substructures of Abs can be prepared by
proteolysis and by recombinant methods. They include the Fab
fragment, which contains the VH-CH1 domains of the heavy chain and
the VL-CL1 domains of the light chain joined by a single interchain
disulfide bond, and the Fv fragment, which contains only the VH and
VL domains. In some cases, a single VH domain retains significant
affinity (Ward et al., 1989). It has also been shown that a certain
monomeric .kappa. light chain will specifically bind to its cognate
antigen. (L. Masat et al., 1994). Separated light or heavy chains
have sometimes been found to retain some antigen-binding activity
(Ward et al., 1989). These antibody fragments are not suitable for
structural analysis using NMR spectroscopy due to their size, low
solubility or low conformational stability.
[0005] Another functional substructure is a single chain Fv (scFv),
made of the variable regions of the immunoglobulin heavy and light
chain, covalently connected by a peptide linker (S-z Hu et al.,
1996). These small (M.sub.r 25,000) proteins generally retain
specificity and affinity for antigen in a single polypeptide and
can provide a convenient building block for larger,
antigen-specific molecules. Several groups have reported
biodistribution studies in xenografted athymic mice using scFv
reactive against a variety of tumor antigens, in which specific
tumor localization has been observed. However, the short
persistence of scFvs in the circulation limits the exposure of
tumor cells to the scFvs, placing limits on the level of uptake. As
a result, tumor uptake by scFvs in animal studies has generally
been only 1-5% ID/g as opposed to intact antibodies that can
localize in tumors ad 30-40% ID/g and have reached levels as high
as 60-70% ID/g.
[0006] A small protein scaffold called a "minibody" was designed
using a part of the Ig VH domain as the template (Pessi et al.,
1993). Minibodies with high affinity (dissociation constant
(K.sub.d).about.10.sup.-7M) to interleukin-6 were identified by
randomizing loops corresponding to CDR1 and CDR2 of VH and then
selecting mutants using the phage display method (Martin et al.,
1994). These experiments demonstrated that the essence of the Ab
function could be transferred to a smaller system. However, the
minibody had inherited the limited solubility of the VH domain
(Bianchi et al., 1994).
[0007] It has been reported that camels (Camelus dromedarius) often
lack variable light chain domains when IgG-like material from their
serum is analyzed, suggesting that sufficient antibody specificity
and affinity can be derived form VH domains (three CDR loops)
alone. Davies and Riechmann recently demonstrated that "camelized"
VH domains with high affinity (K.sub.d.about.10.sup.-7 M) and high
specificity can be generated by randomizing only the CDR3. To
improve the solubility and suppress nonspecific binding, three
mutations were introduced to the framework region (Davies &
Riechmann, 1995). It has not been definitively shown, however, that
camelization can be used, in general, to improve the solubility and
stability of VHs.
[0008] An alternative to the "minibody" is the "diabody." Diabodies
are small bivalent and bispecific antibody fragments, i.e., they
have two antigen-binding sites. The fragments contain a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) on the same polypeptide chain (V.sub.H-V.sub.L).
Diabodies are similar in size to an Fab fragment. By using a linker
that is too short to allow pairing between the two domains on the
same chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
These dimeric antibody fragments, or "diabodies," are bivalent and
bispecific (P. Holliger et al., 1993).
[0009] Since the development of the monoclonal antibody technology,
a large number of 3D structures of Ab fragments in the complexed
and/or free states have been solved by X-ray crystallography
(Webster et al., 1994; Wilson & Stanfield, 1994). Analysis of
Ab structures has revealed that five out of the six CDRs have
limited numbers of peptide backbone conformations, thereby
permitting one to predict the backbone conformation of CDRs using
the so-called canonical structures (Lesk & Tramontano, 1992;
Rees et al., 1994). The analysis also has revealed that the CDR3 of
the VH domain (VH-CDR3) usually has the largest contact surface and
that its conformation is too diverse for canonical structures to be
defined; VH-CDR3 is also known to have a large variation in length
(Wu et al., 1993). Therefore, the structures of crucial regions of
the Ab-antigen interface still need to be experimentally
determined.
[0010] Comparison of crystal structures between the free and
complexed states has revealed several types of conformational
rearrangements. They include side-chain rearrangements, segmental
movements, large rearrangements of VH-CDR3 and changes in the
relative position of the VH and VL domains (Wilson & Stanfield,
1993). In the free state, CDRs, in particular those which undergo
large conformational changes upon binding, are expected to be
flexible. Since X-ray crystallography is not suited for
characterizing flexible parts of molecules, structural studies in
the solution state have not been possible to provide dynamic
pictures of the conformation of antigen-binding sites.
Mimicking the Antibody-Binding Site
[0011] CDR peptides and organic CDR mimetics have been made
(Dougall et al., 1994). CDR peptides are short, typically cyclic,
peptides which correspond to the amino acid sequences of CDR loops
of antibodies. CDR loops are responsible for antibody-antigen
interactions. Organic CDR mimetics are peptides corresponding to
CDR loops which are attached to a scaffold, e.g., a small organic
compound.
[0012] CDR peptides and organic CDR mimetics have been shown to
retain some binding affinity (Smyth & von Itzstein, 1994).
However, as expected, they are too small and too flexible to
maintain full affinity and specificity. Mouse CDRs have been
grafted onto the human Ig framework without the loss of affinity
(Jones et al., 1986; Riechmann et al., 1988), though this
"humanization" does not solve the above-mentioned problems specific
to solution studies.
Mimicking Natural Selection Processes of Abs
[0013] In the immune system, specific Abs are selected and
amplified from a large library (affinity maturation). The processes
can be reproduced in vitro using combinatorial library
technologies. The successful display of Ab fragments on the surface
of bacteriophage has made it possible to generate and screen a vast
number of CDR mutations (McCafferty et al., 1990; Barbas et al.,
1991; Winter et al., 1994). An increasing number of Fabs and Fvs
(and their derivatives) is produced by this technique, providing a
rich source for structural studies. The combinatorial technique can
be combined with Ab mimics.
[0014] A number of protein domains that could potentially serve as
protein scaffolds have been expressed as fusions with phage capsid
proteins. Review in Clackson & Wells, Trends Biotechnol.
12:173-184 (1994). Indeed, several of these protein domains have
already been used as scaffolds for displaying random peptide
sequences, including bovine pancreatic trypsin inhibitor (Roberts
et al., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et
al., Biochemistry 30:10832-10838 (1991)), Venturini et al., Protein
Peptide Letters 1:70-75 (1994)), and the IgG binding domain of
Streptococcus (O'Neil et al., Techniques in Protein Chemistry V
(Crabb, L., ed.) pp. 517-524, Academic Press, San Diego (1994)).
These scaffolds have displayed a single randomized loop or
region.
[0015] Researchers have used the small 74 amino acid
.alpha.-amylase inhibitor Tendamistat as a presentation scaffold on
the filamentous phage M13 (McConnell and Hoess, 1995). Tendamistat
is a .beta.-sheet protein from Streptomyces tendae. It has a number
of features that make it an attractive scaffold for peptides,
including its small size, stability, and the availability of high
resolution NMR and X-ray structural data. Tendamistat's overall
topology is similar to that of an immunoglobulin domain, with two
.beta.-sheets connected by a series of loops. In contrast to
immunoglobulin domains, the .beta.-sheets of Tendamistat are held
together with two rather than one disulfide bond, accounting for
the considerable stability of the protein. By analogy with the CDR
loops found in immunoglobulins, the loops the Tendamistat may serve
a similar function and can be easily randomized by in vitro
mutagenesis.
[0016] Tendamistat, however, is derived from Streptomyces tendae.
Thus, while Tendamistat may be antigenic in humans, its small size
may reduce or inhibit its antigenicity. Also, Tendamistat's
stability is uncertain. Further, the stability that is reported for
Tendamistat is attributed to the presence of two disulfide bonds.
Disulfide bonds, however, are a significant disadvantage to such
molecules in that they can be broken under reducing conditions and
must be properly formed in order to have a useful protein
structure. Further, the size of the loops in Tendamistat are
relatively small, thus limiting the size of the inserts that can be
accommodated in the scaffold. Moreover, it is well known that
forming correct disulfide bonds in newly synthesized peptides is
not straightforward. When a protein is expressed in the cytoplasmic
space of E. coli, the most common host bacterium for protein
overexpression, disulfide bonds are usually not formed, potentially
making it difficult to prepare large quantities of engineered
molecules.
[0017] Thus, there is an on-going need for small, single-chain
artificial antibodies for a variety of therapeutic, diagnostic and
catalytic applications. In particular, there is an on-going need
for artificial antibodies that are structurally stable at neutral
pH.
SUMMARY OF THE INVENTION
[0018] The present invention provides a fibronectin type III (Fn3)
molecule, wherein the Fn3 contains a stabilizing mutation. A
stabilizing mutation is defined herein as a modification or change
in the amino acid sequence of the Fn3 molecule, such as a
substitution of one amino acid for another, that increases the
melting point of the molecule by more than 0.1.degree. C. as
compared to a molecule that is identical except for the change.
Alternatively, the change may increase the melting point by more
than 0.5.degree. C. or even 1.0.degree. C. or more. A method for
determining the melting point of Fn3 molecules is given in Example
19 below.
[0019] The Fn3 may have at least one aspartic acid (Asp) residue
and/or at least one glutamic acid (Glu) residue that has been
deleted or substituted with at least one other amino acid residue.
For example, Asp 7 and/or Asp 23 and/or Glu 9, may have been
deleted or substituted with at least one other amino acid residue.
Asp 7, Asp 23, or Glu 9, may have been substituted with an
asparagine (Asn) or lysine (Lys) residue. The present invention
further provides an isolated nucleic acid molecule and an
expression vector encoding an Fn3 molecule wherein the Fn3 contains
a stabilizing mutation.
[0020] The invention provides a fibronectin type III (Fn3)
polypeptide monobody containing a plurality of Fn3 .beta.-strand
domain sequences that are linked to a plurality of loop region
sequences wherein the Fn3 contains a stabilizing mutation. One or
more of the monobody loop region sequences of the Fn3 polypeptide
vary by deletion, insertion or replacement of at least two amino
acids from the corresponding loop region sequences in wild-type
Fn3. The .beta.-strand domains of the monobody have at least about
50% total amino acid sequence homology to the corresponding amino
acid sequence of wild-type Fn3's .beta.-strand domain sequences.
Preferably, one or more of the loop regions of the monobody contain
amino acid residues:
[0021] i) from 15 to 16 inclusive in an AB loop;
[0022] ii) from 22 to 30 inclusive in a BC loop;
[0023] iii) from 39 to 45 inclusive in a CD loop;
[0024] iv) from 51 to 55 inclusive in a DE loop;
[0025] v) from 60 to 66 inclusive in an EF loop; and
[0026] vi) from 76 to 87 inclusive in an FG loop.
[0027] The invention also provides a nucleic acid molecule encoding
a Fn3 polypeptide monobody wherein the Fn3 contains a stabilizing
mutation, as well as an expression vector containing the nucleic
acid molecule and a host cell containing the vector.
[0028] The invention further provides a method of preparing a Fn3
polypeptide monobody wherein the Fn3 contains a stabilizing
mutation. The method includes providing a DNA sequence encoding a
plurality of Fn3 .beta.-strand domain sequences that are linked to
a plurality of loop region sequences, wherein at least one loop
region of the sequence contains a unique restriction enzyme site.
The DNA sequence is cleaved at the unique restriction site. Then a
preselected DNA segment is inserted into the restriction site. The
preselected DNA segment encodes a peptide capable of binding to a
specific binding partner (SBP) or a transition state analog
compound (TSAC). The insertion of the preselected DNA segment into
the DNA sequence yields a DNA molecule which encodes a polypeptide
monobody having an insertion. The DNA molecule is then expressed so
as to yield the polypeptide monobody.
[0029] Also provided is a method of preparing a Fn3 polypeptide
monobody wherein the Fn3 contains a stabilizing mutation, which
method includes providing a replicatable DNA sequence encoding a
plurality of Fn3 .beta.-strand domain sequences that are linked to
a plurality of loop region sequences, wherein the nucleotide
sequence of at least one loop region is known. Polymerase chain
reaction (PCR) primers are provided or prepared which are
sufficiently complementary to the known loop sequence so as to be
hybridizable under PCR conditions, wherein at least one of the
primers contains a modified nucleic acid sequence to be inserted
into the DNA sequence. PCR is performed using the replicatable DNA
sequence and the primers. The reaction product of the PCR is then
expressed so as to yield a polypeptide monobody.
[0030] The invention provides a further method of preparing a Fn3
polypeptide monobody wherein the Fn3 contains a stabilizing
mutation. The method includes providing a replicatable DNA sequence
encoding a plurality of Fn3 .beta.-strand domain sequences that are
linked to a plurality of loop region sequences, wherein the
nucleotide sequence of at least one loop region is known.
Site-directed mutagenesis of at least one loop region is performed
so as to create an insertion mutation. The resultant DNA including
the insertion mutation is then expressed.
[0031] Further provided is a variegated nucleic acid library
encoding Fn3 polypeptide monobodies including a plurality of
nucleic acid species encoding a plurality of Fn3 .beta.-strand
domain sequences that are linked to a plurality of loop region
sequences, wherein one or more of the monobody loop region
sequences vary by deletion, insertion or replacement of at least
two amino acids from corresponding loop region sequences in
wild-type Fn3, and wherein the .beta.-strand domains of the
monobody have at least a 50% total amino acid sequence homology to
the corresponding amino acid sequence of .beta.-strand domain
sequences of the wild-type Fn3, and wherein the Fn3 contains a
stabilizing mutation. The invention also provides a peptide display
library derived from the variegated nucleic acid library of the
invention. Preferably, the peptide of the peptide display library
is displayed on the surface of a bacteriophage, e.g., a M13
bacteriophage or a fd bacteriophage, or virus.
[0032] The invention also provides a method of identifying the
amino acid sequence of a polypeptide molecule capable of binding to
a specific binding partner (SBP) so as to form a polypeptide:SSP
complex, wherein the dissociation constant of the polypeptide:SBP
complex is less than 10.sup.-6 moles/liter. The method includes the
steps of [0033] a) providing a peptide display library of the
invention; [0034] b) contacting the peptide display library of (a)
with an immobilized or separable SBP; [0035] c) separating the
peptide:SBP complexes from the free peptides; [0036] d) causing the
replication of the separated peptides of (c) so as to result in a
new peptide display library distinguished from that in (a) by
having a lowered diversity and by being enriched in displayed
peptides capable of binding the SBP; [0037] e) optionally repeating
steps (b), (c), and (d) with the new library of (d); and [0038] f)
determining the nucleic acid sequence of the region encoding the
displayed peptide of a species from (d) and hence deducing the
peptide sequence capable of binding to the SBP.
[0039] The present invention also provides a method of preparing a
variegated nucleic acid library encoding Fn3 polypeptide monobodies
having a plurality of nucleic acid species each including a
plurality of loop regions, wherein the species encode a plurality
of Fn3 .beta.-strand domain sequences that are linked to a
plurality of loop region sequences, wherein one or more of the loop
region sequences vary by deletion, insertion or replacement of at
least two amino acids from corresponding loop region sequences in
wild-type Fn3, and wherein the .beta.-strand domain sequences of
the monobody have at least a 50% total amino acid sequence homology
to the corresponding amino acid sequences of .beta.-strand domain
sequences of the wild-type Fn3, and wherein the Fn3 contains a
stabilizing mutation, including the steps of [0040] a) preparing an
Fn3 polypeptide monobody having a predetermined sequence; [0041] b)
contacting the polypeptide with a specific binding partner (SBP) so
as to form a polypeptide:SSP complex wherein the dissociation
constant of the polypeptide:SBP complex is less than 10.sup.-6
moles/liter; [0042] c) determining the binding structure of the
polypeptide:SBP complex by nuclear magnetic resonance spectroscopy
or X-ray crystallography; and [0043] d) preparing the variegated
nucleic acid library, wherein the variegation is performed at
positions in the nucleic acid sequence which, from the information
provided in (c), result in one or more polypeptides with improved
binding to the SBP.
[0044] Also provided is a method of identifying the amino acid
sequence of a polypeptide molecule capable of catalyzing a chemical
reaction with a catalyzed rate constant, k.sub.cat, and an
uncatalyzed rate constant, k.sub.uncat, such that the ratio of
k.sub.cat/k.sub.uncat greater than 10. The method includes the
steps of: [0045] a) providing a peptide display library of the
invention; [0046] b) contacting the peptide display library of (a)
with an immobilized or separable transition state analog compound
(TSAC) representing the approximate molecular transition state of
the chemical reaction; [0047] c) separating the peptide:TSAC
complexes from the free peptides; [0048] d) causing the replication
of the separated peptides of (c) so as to result in a new peptide
display library distinguished from that in (a) by having a lowered
diversity and by being enriched in displayed peptides capable of
binding the TSAC; [0049] e) optionally repeating steps (b), (c),
and (d) with the new library of (d); and [0050] f) determining the
nucleic acid sequence of the region encoding the displayed peptide
of a species from (d) and hence deducing the peptide sequence.
[0051] The invention also provides a method of preparing a
variegated nucleic acid library encoding Fn3 polypeptide monobodies
having a plurality of nucleic acid species each including a
plurality of loop regions, wherein the species encode a plurality
of Fn3 .beta.-strand domain sequences that are linked to a
plurality of loop region sequences, wherein one or more of the loop
region sequences vary by deletion, insertion or replacement of at
least two amino acids from corresponding loop region sequences in
wild-type Fn3, and wherein the .beta.-strand domain sequences of
the monobody have at least a 50% total amino acid sequence homology
to the corresponding amino acid sequences of .beta.-strand domain
sequences of the wild-type Fn3, and wherein the Fn3 contains a
stabilizing mutation, including the steps of [0052] a) preparing an
Fn3 polypeptide monobody having a predetermined sequence, wherein
the polypeptide is capable of catalyzing a chemical reaction with a
catalyzed rate constant, k.sub.cat, and an uncatalyzed rate
constant, k.sub.uncat, such that the ratio of k.sub.cat/k.sub.uncat
is greater than 10; [0053] b) contacting the polypeptide with an
immobilized or separable transition state analog compound (TSAC)
representing the approximate molecular transition state of the
chemical reaction; [0054] c) determining the binding structure of
the polypeptide:TSAC complex by nuclear magnetic resonance
spectroscopy or X-ray crystallography; and [0055] d) preparing the
variegated nucleic acid library, wherein the variegation is
performed at positions in the nucleic acid sequence which, from the
information provided in (c), result in one or more polypeptides
with improved binding to or stabilization of the TSAC.
[0056] The invention also provides a kit for the performance of any
of the methods of the invention. The invention further provides a
composition, e.g., a polypeptide, prepared by the use of the kit,
or identified by any of the methods of the invention.
[0057] The following abbreviations have been used in describing
amino acids, peptides, or proteins: Ala or A, Alanine; Arg or R,
Arginine; Asn or N asparagine; Asp or D, aspartic acid; Cys or C,
cysteine; Gln or Q, glutamine; Glu or E, glutamic acid; Gly or G,
glycine; His or H, histidine; Ile or I, isoleucine; Leu or L,
leucine; Lys or K, lysine; Met or M, methionine; Phe or F,
phenylalanine; Pro or P, proline; Ser or S, serine; Thr or T,
threonine; Trp or W, tryptophan; Tyr or Y, tyrosine; Val or V,
valine.
[0058] The following abbreviations have been used in describing
nucleic acids, DNA, or RNA: A, adenosine; T, thymidine; G,
guanosine; C, cytosine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1. .beta.-Strand and loop topology (A, B) and MOLSCRIPT
representation (C, D; Kraulis, 1991) of the VH domain of
anti-lysozyme immunoglobulin D1.3 (A, C; Bhat et al., 1994) and
10th type III domain of human fibronectin (B, D; Main et al.,
1992). The locations of complementarity determining regions (CDRs,
hypervariable regions) and the integrin-binding Arg-Gly-Asp (RGD)
sequence are indicated.
[0060] FIG. 2. Amino acid sequence (SEQ ID NO:110) and restriction
sites of the synthetic Fn3 gene. The residue numbering is according
to Main et al. (1992). Restriction enzyme sites designed are shown
above the amino acid sequence. .beta.-Strands are denoted by
underlines. The N-terminal "mq" sequence has been added for a
subsequent cloning into an expression vector. The His.tag (Novagen)
fusion protein has an additional sequence, MGSSHHHHHHSSGLVPRGSH
(SEQ ID NO:114), preceding the Fn3 sequence shown above.
[0061] FIG. 3. A, Far UV CD spectra of wild-type Fn3 at 25.degree.
C. and 90.degree. C. Fn3 (50 .mu.M) was dissolved in sodium acetate
(50 mM, pH 4.6). B, thermal denaturation of Fn3 monitored at 215
nm. Temperature was increased at a rate of 1.degree. C./min.
[0062] FIG. 4. A, C.alpha. trace of the crystal structure of the
complex of lysozyme (HEL) and the Fv fragment of the anti-hen
egg-white lysozyme (anti-HEL) antibody D1.3 (Bhat et al., 1994).
Side chains of the residues 99-102 of VH CDR3, which make contact
with HEL, are also shown. B, Contact surface area for each residue
of the D1.3 VH-HEL and VH-VL interactions plotted vs. residue
number of D1.3 VH. Surface area and secondary structure were
determined using the program DSSP (Kabsh and Sander, 1983). C and
D, schematic drawings of the .beta.-sheet structure of the F
strand-loop-G strand moieties of D1.3 VH (C) and Fn3 (D). The boxes
denote residues in .beta.-strands and ovals those not in strands.
The shaded boxes indicate residues of which side chains are
significantly buried. The broken lines indicate hydrogen bonds.
[0063] FIG. 5. Designed Fn3 gene showing DNA (SEQ ID NO:111) and
amino acid (SEQ ID NO:112) sequences. The amino acid numbering is
according to Main et al. (1992). The two loops that were randomized
in combinatorial libraries are enclosed in boxes.
[0064] FIG. 6. Map of plasmid pAS45. Plasmid pAS45 is the
expression vector of His.tag-Fn3.
[0065] FIG. 7. Map of plasmid pAS25. Plasmid pAS25 is the
expression vector of Fn3.
[0066] FIG. 8. Map of plasmid pAS38. pAS38 is a phagemid vector for
the surface display of Fn3.
[0067] FIG. 9. (Ubiquitin-1) Characterization of ligand-specific
binding of enriched clones using phage enzyme-linked immunosolvent
assay (ELISA). Microtiter plate wells were coated with ubiquitin (1
.mu.g/well; "Ligand (+)) and then blocked with BSA. Phage solution
in TBS containing approximately 10.sup.10 colony forming units
(cfu) was added to a well and washed with TBS. Bound phages were
detected with anti-phage antibody-POD conjugate (Pharmacia) with
Turbo-TMB (Pierce) as a substrate. Absorbance was measured using a
Molecular Devices SPECTRAmax 250 microplate spectrophotometer. For
a control, wells without the immobilized ligand were used. 2-1 and
2-2 denote enriched clones from Library 2 eluted with free ligand
and acid, respectively. 4-1 and 4-2 denote enriched clones from
Library 4 eluted with free ligand and acid, respectively.
[0068] FIG. 10. (Ubiquitin-2) Competition phage ELISA of enriched
clones.
[0069] Phage solutions containing approximately 10.sup.10 cfu were
first incubated with free ubiquitin at 4.degree. C. for 1 hour
prior to the binding to a ligand-coated well. The wells were washed
and phages detected as described above.
[0070] FIG. 11. Competition phage ELISA of ubiquitin-binding
monobody 411. Experimental conditions are the same as described
above for ubiquitin. The ELISA was performed in the presence of
free ubiquitin in the binding solution. The experiments were
performed with four different preparations of the same clone.
[0071] FIG. 12. (Fluorescein-1) Phage ELISA of four clones, Plb25.1
(containing SEQ ID NO:115), Plb25.4 (containing SEQ ID NO:116),
pLB24.1 (containing SEQ ID NO:117) and pLB24.3 (containing SEQ ID
NO:118). Experimental conditions are the same as ubiquitin-1
above.
[0072] FIG. 13. (Fluorescein-2) Competition ELISA of the four
clones. Experimental conditions are the same as ubiquitin-2
above.
[0073] FIG. 14. .sup.1H, .sup.15N-HSQC spectrum of a
fluorescence-binding monobody LB25.5. Approximately 20 .mu.M
protein was dissolved in 10 mM sodium acetate buffer (pH 5.0)
containing 100 mM sodium chloride. The spectrum was collected at
30.degree. C. on a Varian Unity INOVA 600 NMR spectrometer.
[0074] FIG. 15. Characterization of the binding reaction of
Ubi4-Fn3 to the target, ubiquitin. (a) Phage ELISA analysis of
binding of Ubi4-Fn3 to ubiquitin. The binding of Ubi4-phages to
ubiquitin-coated wells was measured. The control experiment was
performed with wells containing no ubiquitin.
[0075] (b) Competition phage ELISA of Ubi4-Fn3. Ubi4-Fn3-phages
were preincubated with soluble ubiquitin at an indicated
concentration, followed by the phage ELISA detection in
ubiquitin-coated wells.
[0076] (c) Competition phage ELISA testing the specificity of the
Ubi4 clone. The Ubi4 phages were preincubated with 250 .mu.g/ml of
soluble proteins, followed by phage ELISA as in (b).
[0077] (d) ELISA using free proteins.
[0078] FIG. 16. Equilibrium unfolding curves for Ubi4-Fn3 (closed
symbols) and wild-type Fn3 (open symbols). Squares indicate data
measured in TBS (Tris HCl buffer (50 mM, pH 7.5) containing NaCl
(150 mM)). Circles indicate data measured in Gly HCl buffer (20 mM,
pH 3.3) containing NaCl (300 mM). The curves show the best fit of
the transition curve based on the two-state model. Parameters
characterizing the transitions are listed in Table 8.
[0079] FIG. 17. (a) .sup.1H, .sup.15N-HSQC spectrum of
[.sup.15N]-Ubi4-K Fn3. (b). Difference
(.delta..sub.wild-type-.delta..sub.Ubi4) of .sup.1H (b) and
.sup.15N (c) chemical shifts plotted versus residue number. Values
for residues 82-84 (shown as filled circles) where Ubi4-K deletions
are set to zero. Open circles indicate residues that are mutated in
the Ubi4-K protein. The locations of .beta.-strands are indicated
with arrows.
[0080] FIG. 18. (A) Guanidine hydrochloride (GuHCl)-induced
denaturation of FNfn10 monitored by Trp fluorescence. The
fluorescence emission intensity at 355 nm is shown as a function of
GuHCl concentration. The lines show the best fits of the data to
the two-state transition model. (B) Stability of FN3 at 4 M GuHCl
plotted as a function of pH. (C) pH dependence of the m value.
[0081] FIG. 19. A two-dimensional H(C)CO spectrum of FNfn10 showing
the .sup.13C chemical shift of the carboxyl carbon (vertical axis)
and the .sup.1H shift of .sup.1H.sup..beta. of Asp or
.sup.1H.sup..gamma. of Glu, respectively (horizontal axis). Cross
peaks are labeled with their respective residue numbers.
[0082] FIG. 20. pH-Dependent shifts of the .sup.13C chemical shifts
of the carboxyl carbons of Asp and Glu residues in FNfn10. Panel A
shows data for Asp 3, 67 and 80, and Glu 38 and 47. The lines are
the best fits of the data to the Henderson-Hasselbalch equation
with one ionizable group (McIntosh, L. P., Hand, G., Johnson, P.
E., Joshi, M. D., Koerner, M., Plesniak, L. A., Ziser, L.,
Wakarchuk, W. W. & Withers, S. G. (1996) Biochemistry 35,
9958-9966). Panel B shows data for Asp 7 and 23 and Glu 9. The
continuous lines show the best fits to the Henderson-Hasselbalch
equation with two ionizable groups, while the dashed lines show the
best fits to the equation with a single ionizable group.
[0083] FIG. 21. (A) The amino acid sequence of FNfn10 (SEQ ID
NO:121) shown according to its topology (Main, A. L., Harvey, T.
S., Baron, M., Boyd, J., & Campbell, I. D. (1992) Cell 71,
671-678). Asp and Glu residues are highlighted with gray circles.
The thin lines and arrows connecting circles indicate backbone
hydrogen bonds. (B) A CPK model of FN3 showing the locations of Asp
7 and 23 and Glu 9.
[0084] FIG. 22. Thermal denaturation of the wild-type and mutant
FNfn10 proteins at pH 7.0 and 2.4 in the presence of 6.3 M urea and
0.1 or 1.0 M NaCl. Change in circular dichroism signal at 227 nm is
plotted as a function of temperature. The filled circles show the
data in the presence of 1 M NaCl and the open circles are data in
the presence of 0.1 M NaCl. The left column shows data taken at pH
2.4 and the right column at pH 7.0. The identity of proteins is
indicated in the panels.
[0085] FIG. 23. GuHCl-induce denaturation of FNfn10 mutants
monitored with fluorescence. Fluorescence data was converted to the
fraction of unfolded protein according to the two-state transition
model (Loladze, V. V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. &
Makhatadze, G. I. (1999) Biochemistry 38, 16419-16423), and plotted
as a function of GuHCl.
[0086] FIG. 24. pH Titration of the carboxyl .sup.13C resonance of
Asp and Glu residues in D7N (open circles) and D7K (closed circles)
FNfn10. Data for the wild-type (crosses) are also shown for
comparison. Residue names are denoted in the individual panels.
DETAILED DESCRIPTION OF THE INVENTION
[0087] For the past decade the immune system has been exploited as
a rich source of de novo catalysts. Catalytic antibodies have been
shown to have chemoselectivity, enantioselectivity, large rate
accelerations, and even an ability to reroute chemical reactions.
In most cases the antibodies have been elicited to transition state
analog (TSA) haptens. These TSA haptens are stable, low-molecular
weight compounds designed to mimic the structures of the
energetically unstable transition state species that briefly
(approximate half-life 10.sup.-13 s) appear along reaction pathways
between reactants and products. Anti-TSA antibodies, like natural
enzymes, are thought to selectively bind and stabilize transition
state, thereby easing the passage of reactants to products. Thus,
upon binding, the antibody lowers the energy of the actual
transition state and increases the rate of the reaction. These
catalysts can be programmed to bind to geometrical and
electrostatic features of the transition state so that the reaction
route can be controlled by neutralizing unfavorable charges,
overcoming entropic barriers, and dictating stereoelectronic
features of the reaction. By this means even reactions that are
otherwise highly disfavored have been catalyzed (Janda et al.
1997). Further, in many instances catalysts have been made for
reactions for which there are no known natural or man-made
enzymes.
[0088] The success of any combinatorial chemical system in
obtaining a particular function depends on the size of the library
and the ability to access its members. Most often the antibodies
that are made in an animal against a hapten that mimics the
transition state of a reaction are first screened for binding to
the hapten and then screened again for catalytic activity. An
improved method allows for the direct selection for catalysis from
antibody libraries in phage, thereby linking chemistry and
replication.
[0089] A library of antibody fragments can be created on the
surface of filamentous phage viruses by adding randomized antibody
genes to the gene that encodes the phage's coat protein. Each phage
then expresses and displays multiple copies of a single antibody
fragment on its surface. Because each phage possesses both the
surface-displayed antibody fragment and the DNA that encodes that
fragment, and antibody fragment that binds to a target can be
identified by amplifying the associated DNA.
[0090] Immunochemists use as antigens materials that have as little
chemical reactivity as possible. It is almost always the case that
one wishes the ultimate antibody to interact with native
structures. In reactive immunization the concept is just the
opposite. One immunizes with compounds that are highly reactive so
that upon binding to the antibody molecule during the induction
process, a chemical reaction ensues. Later this same chemical
reaction becomes part of the mechanism of the catalytic event. In a
certain sense one is immunizing with a chemical reaction rather
than a substance per se. Reactive immunogens can be considered as
analogous to the mechanism-based inhibitors that enzymologists use
except that they are used in the inverse way in that, instead of
inhibiting a mechanism, they induce a mechanism.
[0091] Man-made catalytic antibodies have considerable commercial
potential in many different applications. Catalytic antibody-based
products have been used successfully in prototype experiments in
therapeutic applications, such as prodrug activation and cocaine
inactivation, and in nontherapeutic applications, such as
biosensors and organic synthesis.
[0092] Catalytic antibodies are theoretically more attractive than
noncatalytic antibodies as therapeutic agents because, being
catalytic, they may be used in lower doses, and also because their
effects are unusually irreversible (for example, peptide bond
cleavage rather than binding). In therapy, purified catalytic
antibodies could be directly administered to a patient, or
alternatively the patient's own catalytic antibody response could
be elicited by immunization with an appropriate hapten. Catalytic
antibodies also could be used as clinical diagnostic tools or as
regioselective or stereoselective catalysts in the synthesis of
fine chemicals.
I. Mutation of Fn3 Loops and Grafting of Ab Loops onto Fn3
[0093] An ideal scaffold for CDR grafting is highly soluble and
stable. It is small enough for structural analysis, yet large
enough to accommodate multiple CDRs so as to achieve tight binding
and/or high specificity.
[0094] A novel strategy to generate an artificial Ab system on the
framework of an existing non-Ab protein was developed. An advantage
of this approach over the minimization of an Ab scaffold is that
one can avoid inheriting the undesired properties of Abs.
Fibronectin type III domain (Fn3) was used as the scaffold.
Fibronectin is a large protein which plays essential roles in the
formation of extracellular matrix and cell-cell interactions; it
consists of many repeats of three types (I, II and III) of small
domains (Baron et al., 1991). Fn3 itself is the paradigm of a large
subfamily (Fn3 family or s-type Ig family) of the immunoglobulin
superfamily (IgSF). The Fn3 family includes cell adhesion
molecules, cell surface hormone and cytokine receptors,
chaperonins, and carbohydrate-binding domains (for reviews, see
Bork & Doolittle, 1992; Jones, 1993; Bork et al., 1994;
Campbell & Spitzfaden, 1994; Harpez & Chothia, 1994).
[0095] Recently, crystallographic studies revealed that the
structure of the DNA binding domains of the transcription factor
NF-kB is also closely related to the Fn3 fold (Ghosh et al., 1995;
Muller et al., 1995). These proteins are all involved in specific
molecular recognition, and in most cases ligand-binding sites are
formed by surface loops, suggesting that the Fn3 scaffold is an
excellent framework for building specific binding proteins. The 3D
structure of Fn3 has been determined by NMR (Main et al., 1992) and
by X-ray crystallography (Leahy et al., 1992; Dickinson et al.,
1994). The structure is best described as a .beta.-sandwich similar
to that of Ab VH domain except that Fn3 has seven .beta.-strands
instead of nine (FIG. 1). There are three loops on each end of Fn3;
the positions of the BC, DE and FG loops approximately correspond
to those of CDR1, 2 and 3 of the VH domain, respectively (FIG. 1 C,
D).
[0096] Fn3 is small (.about.95 residues), monomeric, soluble and
stable. It is one of few members of IgSF that do not have disulfide
bonds; VH has an interstrand disulfide bond (FIG. 1 A) and has
marginal stability under reducing conditions. Fn3 has been
expressed in E. coli (Aukhil et al., 1993). In addition, 17 Fn3
domains are present just in human fibronectin, providing important
information on conserved residues which are often important for the
stability and folding (for sequence alignment, see Main et al.,
1992 and Dickinson et al., 1994). From sequence analysis, large
variations are seen in the BC and FG loops, suggesting that the
loops are not crucial to stability. NMR studies have revealed that
the FG loop is highly flexible; the flexibility has been implicated
for the specific binding of the 10th Fn3 to
.alpha..sub.5.beta..sub.1 integrin through the Arg-Gly-Asp (RGD)
motif. In the crystal structure of human growth hormone-receptor
complex (de Vos et al., 1992), the second Fn3 domain of the
receptor interacts with hormone via the FG and BC loops, suggesting
it is feasible to build a binding site using the two loops.
[0097] The tenth type III module of fibronectin has a fold similar
to that of immunoglobulin domains, with seven .beta. strands
forming two antiparallel .beta. sheets, which pack against each
other (Main et al., 1992). The structure of the type II module
consists of seven .beta. strands, which form a sandwich of two
antiparallel .beta. sheets, one containing three strands (ABE) and
the other four strands (C'CFG) (Williams et al., 1988). The
triple-stranded .beta. sheet consists of residues Glu-9-Thr-14 (A),
Ser-17-Asp-23 (B), and Thr-56-Ser-60 (E). The majority of the
conserved residues contribute to the hydrophobic core, with the
invariant hydrophobic residues Trp-22 and Try-68 lying toward the
N-terminal and C-terminal ends of the core, respectively. The
.beta. strands are much less flexible and appear to provide a rigid
framework upon which functional, flexible loops are built. The
topology is similar to that of immunoglobulin C domains.
Gene Construction and Mutagenesis
[0098] A synthetic gene for tenth Fn3 of human fibronectin (FIG. 2)
was designed which includes convenient restriction sites for ease
of mutagenesis and uses specific codons for high-level protein
expression (Gribskov et al., 1984).
[0099] The gene was assembled as follows: (1) the gene sequence was
divided into five parts with boundaries at designed restriction
sites (FIG. 2); (2) for each part, a pair of oligonucleotides that
code opposite strands and have complementary overlaps of .about.15
bases was synthesized; (3) the two oligonucleotides were annealed
and single strand regions were filled in using the Klenow fragment
of DNA polymerase; (4) the double-stranded oligonucleotide was
cloned into the pET3a vector (Novagen) using restriction enzyme
sites at the termini of the fragment and its sequence was confirmed
by an Applied Biosystems DNA sequencer using the dideoxy
termination protocol provided by the manufacturer; (5) steps 2-4
were repeated to obtain the whole gene (plasmid pAS25) (FIG.
7).
[0100] Although the present method takes more time to assemble a
gene than the one-step polymerase chain reaction (PCR) method
(Sandhu et al., 1992), no mutations occurred in the gene. Mutations
would likely have been introduced by the low fidelity replication
by Taq polymerase and would have required time-consuming gene
editing. The gene was also cloned into the pET15b (Novagen) vector
(pEW1). Both vectors expressed the Fn3 gene under the control of
bacteriophage T7 promoter (Studler et al. 1990); pAS25 expressed
the 96-residue Fn3 protein only, while pEW1 expressed Fn3 as a
fusion protein with poly-histidine peptide (His.tag). Recombinant
DNA manipulations were performed according to Molecular Cloning
(Sambrook et al., 1989), unless otherwise stated.
[0101] Mutations were introduced to the Fn3 gene using either
cassette mutagenesis or oligonucleotide site-directed mutagenesis
techniques (Deng & Nickoloff, 1992). Cassette mutagenesis was
performed using the same protocol for gene construction described
above; double-stranded DNA fragment coding a new sequence was
cloned into an expression vector (pAS25 and/or pEW1). Many
mutations can be made by combining a newly synthesized strand
(coding mutations) and an oligonucleotide used for the gene
synthesis. The resulting genes were sequenced to confirm that the
designed mutations and no other mutations were introduced by
mutagenesis reactions.
Design and Synthesis of Fn3 Mutants with Antibody CDRs
[0102] Two candidate loops (FG and BC) were identified for
grafting. Antibodies with known crystal structures were examined in
order to identify candidates for the sources of loops to be grafted
onto Fn3. Anti-hen egg lysozyme (HEL) antibody D1.3 (Bhat et al.,
1994) was chosen as the source of a CDR loop. The reasons for this
choice were: (1) high resolution crystal structures of the free and
complexed states are available (FIG. 4 A; Bhat et al., 1994), (2)
thermodynamics data for the binding reaction are available (Tello
et al., 1993), (3) D1.3 has been used as a paradigm for Ab
structural analysis and Ab engineering (Verhoeyen et al., 1988;
McCafferty et al., 1990) (4) site-directed mutagenesis experiments
have shown that CDR3 of the heavy chain (VH-CDR3) makes a larger
contribution to the affinity than the other CDRs (Hawkins et al.,
1993), and (5) a binding assay can be easily performed. The
objective for this trial was to graft VH-CDR3 of D1.3 onto the Fn3
scaffold without significant loss of stability.
[0103] An analysis of the D1.3 structure (FIG. 4) revealed that
only residues 99-102 ("RDYR") (SEQ ID NO:120) make direct contact
with hen egg-white lysozyme (HEL) (FIG. 4 B), although VH-CDR3 is
defined as longer (Bhat et al., 1994). It should be noted that the
C-terminal half of VH-CDR3 (residues 101-104) made significant
contact with the VL domain (FIG. 4 B). It has also become clear
that D1.3 VH-CDR3 (FIG. 4 C) has a shorter turn between the strands
F and G than the FG loop of Fn3 (FIG. 4 D). Therefore, mutant
sequences were designed by using the RDYR (99-102) (SEQ ID NO:120)
of D1.3 as the core and made different boundaries and loop lengths
(Table 1). Shorter loops may mimic the D1.3 CDR3 conformation
better, thereby yielding higher affinity, but they may also
significantly reduce stability by removing wild-type interactions
of Fn3.
TABLE-US-00001 TABLE 1 Amino acid sequences of D1.3 VH CDR3, VH8
CDR3 and Fn3 FG loop and list of planned mutants. D1.3 96 100 105
(SEQ ID NO: 1) .cndot. .cndot. .cndot. ARERDYRLDYWGQG VH8
ARGAVVSYYAMDYWGQG (SEQ ID NO: 2) Fn3 75 80 85 (SEQ ID NO: 3)
.cndot. .cndot. .cndot. YAVTGRGDSPASSKPI Mutant Sequence D1.3-1
YAERDYRLDY----PI (SEQ ID NO: 4) D1.3-2 YAVRDYRLDY----PI (SEQ ID NO:
5) D1.3-3 YAVRDYRLDYASSKPI (SEQ ID NO: 6) D1.3-4 YAVRDYRLDY---KPI
(SEQ ID NO: 7) D1.3-5 YAVRDYR-----SKPI (SEQ ID NO: 8) D1.3-6
YAVTRDYRL--SSKPI (SEQ ID NO: 9) D1.3-7 YAVTERDYRL-SSKPI (SEQ ID NO:
10) VH8-1 YAVAVVSYYAMDY-PI (SEQ ID NO: 11) VH8-2 YAVTAVVSYYASSKPI
(SEQ ID NO: 12) Underlines indicate residues in .beta.-strands.
Bold characters indicate replaced residues.
[0104] In addition, an anti-HEL single VH domain termed VH8 (Ward
et al., 1989) was chosen as a template. VH8 was selected by library
screening and, in spite of the lack of the VL domain, VH8 has an
affinity for HEL of 27 nM, probably due to its longer VH-CDR3
(Table 1). Therefore, its VH-CDR3 was grafted onto Fn3. Longer
loops may be advantageous on the Fn3 framework because they may
provide higher affinity and also are close to the loop length of
wild-type Fn3. The 3D structure of VH8 was not known and thus the
VH8 CDR3 sequence was aligned with that of D1.3 VH-CDR3; two loops
were designed (Table 1).
Mutant Construction and Production
[0105] Site-directed mutagenesis experiments were performed to
obtain designed sequences. Two mutant Fn3s, D1.3-1 and D1.3-4
(Table 1) were obtained and both were expressed as soluble His.tag
fusion proteins. D1.3-4 was purified and the His.tag portion was
removed by thrombin cleavage. D1.3-4 is soluble up to at least 1 mM
at pH 7.2. No aggregation of the protein has been observed during
sample preparation and NMR data acquisition.
Protein Expression and Purification
[0106] E. coli BL21 (DE3) (Novagen) were transformed with an
expression vector (pAS25, pEW1 and their derivatives) containing a
gene for the wild-type or a mutant. Cells were grown in M9 minimal
medium and M9 medium supplemented with Bactotrypton (Difco)
containing ampicillin (200 .mu.g/ml). For isotopic labeling,
.sup.15N NH.sub.4Cl and/or .sup.13C glucose replaced unlabeled
components. 500 ml medium in a 2 liter baffle flask were inoculated
with 10 ml of overnight culture and agitated at 37.degree. C.
Isopropylthio-.beta.-galactoside (IPTG) was added at a final
concentration of 1 mM to initiate protein expression when OD (600
nm) reaches one. The cells were harvested by centrifugation 3 hours
after the addition of IPTG and kept frozen at -70.degree. C. until
used.
[0107] Fn3 without His.tag was purified as follows. Cells were
suspended in 5 ml/(g cell) of Tris (50 mM, pH 7.6) containing
ethylenediaminetetraacetic acid (EDTA; 1 mM) and
phenylmethylsulfonyl fluoride (1 mM). HEL was added to a final
concentration of 0.5 mg/ml. After incubating the solution for 30
minutes at 37.degree. C., it was sonicated three times for 30
seconds on ice. Cell debris was removed by centrifugation. Ammonium
sulfate was added to the solution and precipitate recovered by
centrifugation. The pellet was dissolved in 5-10 ml sodium acetate
(50 mM, pH 4.6) and insoluble material was removed by
centrifugation. The solution was applied to a Sephacryl S100HR
column (Pharmacia) equilibrated in the sodium acetate buffer.
Fractions containing Fn3 then was applied to a ResourceS column
(Pharmacia) equilibrated in sodium acetate (50 mM, pH 4.6) and
eluted with a linear gradient of sodium chloride (0-0.5 M). The
protocol can be adjusted to purify mutant proteins with different
surface charge properties.
[0108] Fn3 with His.tag was purified as follows. The soluble
fraction was prepared as described above, except that sodium
phosphate buffer (50 mM, pH 7.6) containing sodium chloride (100
mM) replaced the Tris buffer. The solution was applied to a Hi-Trap
chelating column (Pharmacia) preloaded with nickel and equilibrated
in the phosphate buffer. After washing the column with the buffer,
His.tag-Fn3 was eluted in the phosphate buffer containing 50 mM
EDTA. Fractions containing His.tag-Fn3 were pooled and applied to a
Sephacryl S100-HR column, yielding highly pure protein. The His.tag
portion was cleaved off by treating the fusion protein with
thrombin using the protocol supplied by Novagen. Fn3 was separated
from the His.tag peptide and thrombin by a ResourceS column using
the protocol above.
[0109] The wild-type and two mutant proteins so far examined are
expressed as soluble proteins. In the case that a mutant is
expressed as inclusion bodies (insoluble aggregate), it is first
examined if it can be expressed as a soluble protein at lower
temperature (e.g., 25-30.degree. C.). If this is not possible, the
inclusion bodies are collected by low-speed centrifugation
following cell lysis as described above. The pellet is washed with
buffer, sonicated and centrifuged. The inclusion bodies are
solubilized in phosphate buffer (50 mM, pH 7.6) containing
guanidinium chloride (GdnCl, 6 M) and will be loaded on a Hi-Trap
chelating column. The protein is eluted with the buffer containing
GdnCl and 50 mM EDTA.
Conformation of Mutant Fn3, D1.3-4
[0110] The .sup.1H NMR spectra of His.tag D1.3-4 fusion protein
closely resembled that of the wild-type, suggesting the mutant is
folded in a similar conformation to that of the wild-type. The
spectrum of D1.3-4 after the removal of the His.tag peptide showed
a large spectral dispersion. A large dispersion of amide protons
(7-9.5 ppm) and a large number of downfield (5.0-6.5 ppm)
C.sup..alpha. protons are characteristic of a .beta.-sheet protein
(Wuthrich, 1986).
[0111] The 2D NOESY spectrum of D1.3-4 provided further evidence
for a preserved conformation. The region in the spectrum showed
interactions between upfield methyl protons (<0.5 ppm) and
methyl-methylene protons. The Val72 .gamma. methyl resonances were
well separated in the wild-type spectrum (-0.07 and 0.37 ppm;
(Baron et al., 1992)). Resonances corresponding to the two methyl
protons are present in the D1.3-4 spectrum (-0.07 and 0.44 ppm).
The cross peak between these two resonances and other conserved
cross peaks indicate that the two resonances in the D1.3-4 spectrum
are highly likely those of Val72 and that other methyl protons are
in nearly identical environment to that of wild-type Fn3. Minor
differences between the two spectra are presumably due to small
structural perturbation due to the mutations. Val72 is on the F
strand, where it forms a part of the central hydrophobic core of
Fn3 (Main et al., 1992). It is only four residues away from the
mutated residues of the FG loop (Table 1). The results are
remarkable because, despite there being 7 mutations and 3 deletions
in the loop (more than 10% of total residues; FIG. 12, Table 2),
D1.3-4 retains a 3D structure virtually identical to that of the
wild-type (except for the mutated loop). Therefore, the results
provide strong support that the FG loop is not significantly
contributing to the folding and stability of the Fn3 molecule and
thus that the FG loop can be mutated extensively.
TABLE-US-00002 TABLE 2 Sequences of oligonucleotides Name Sequence
FN1F CGGGATCCCATATGCAGGTTTCTGATGTTCCGCGTGACC TGGAAGTTGTTGCTGCGACC
(SEQ ID NO: 13) FN1R TAACTGCAGGAGCATCCCAGCTGATCAGCAGGCTAGTC
GGGGTCGCAGCAACAAC (SEQ ID NO: 14) FN2F
CTCCTGCAGTTACCGTGCGTTATTACCGTATCACGTACG GTGAAACCGGTG (SEQ ID NO:
15) FN2R GTGAATTCCTGAACCGGGGAGTTACCACCGGTTTCACC G (SEQ ID NO: 16)
FN3F AGGAATTCACTGTACCTGGTTCCAAGTCTACTGCTACCA TCAGCGG (SEQ ID NO:
17) FN3R GTATAGTCGACACCCGGTTTCAGGCCGCTGATGGTAGC (SEQ ID NO: 18)
FN4F CGGGTGTCGACTATACCATCACTGTATACGCT (SEQ ID NO: 19) FN4R
CGGGATCCGAGCTCGCTGGGCTGTCACCACGGCCAGTA ACAGCGTATACAGTGAT (SEQ ID
NO: 20) FN5F CAGCGAGCTCCAAGCCAATCTCGATTAACTACCGT (SEQ ID NO: 21)
FN5R CGGGATCCTCGAGTTACTAGGTACGGTAGTTAATCGA (SEQ ID NO: 22) FN5R'
CGGGATCCACGCGTGCCACCGGTACGGTAGTTAATCGA (SEQ ID NO: 23) gene3F
CGGGATCCACGCGTCCATTCGTTTGTGAATATCAAGGCC AATCG (SEQ ID NO: 24)
gene3R CCGGAAGCTTTAAGACTCCTTATTACGCAGTATGTTAGC (SEQ ID NO: 25)
38TAABglII CTGTTACTGGCCGTGAGATCTAACCAGCGAGCTCCA (SEQ ID NO: 26) BC3
GATCAGCTGGGATGCTCCTNNKNNKNNKNNKNNKTATT ACCGTATCACGTA (SEQ ID NO:
27) FG2 TGTATACGCTGTTACTGGCNNKNNKNNKNNKNNKNNKN NKTCCAAGCCAATCTCGAT
(SEQ ID NO: 28) FG3 CTGTATACGCTGTTACTGGCNNKNNKNNKNNKCCAGCG
AGCTCCAAG (SEQ ID NO: 29) FG4
CATCACTGTATACGCTGTTACTNNKNNKNNKNNKNNKT CCAAGCCAATCTC (SEQ ID NO:
30) Restriction enzyme sites are underlined. N and K denote an
equimolar mixture of A, T. G and C and that of G and T,
respectively.
Structure and Stability Measurements
[0112] Structures of Abs were analyzed using quantitative methods
(e.g., DSSP (Kabsch & Sander, 1983) and PDBfit (D. McRee, The
Scripps Research Institute)) as well as computer graphics (e.g.,
Quanta (Molecular Simulations) and What if (G. Vriend, European
Molecular Biology Laboratory)) to superimpose the
strand-loop-strand structures of Abs and Fn3.
[0113] The stability of monobodies was determined by measuring
temperature- and chemical denaturant-induced unfolding reactions
(Pace et al., 1989). The temperature-induced unfolding reaction was
measured using a circular dichroism (CD) polarimeter. Ellipticity
at 222 and 215 nm was recorded as the sample temperature was slowly
raised. Sample concentrations between 10 and 50 .mu.M were used.
After the unfolding baseline was established, the temperature was
lowered to examine the reversibility of the unfolding reaction.
Free energy of unfolding was determined by fitting data to the
equation for the two-state transition (Becktel & Schellman,
1987; Pace et al., 1989). Nonlinear least-squares fitting was
performed using the program Igor (WaveMetrics) on a Macintosh
computer.
[0114] The structure and stability of two selected mutant Fn3s were
studied; the first mutant was D1.3-4 (Table 2) and the second was a
mutant called AS40 which contains four mutations in the BC loop
(A.sup.26V.sup.27T.sup.28V.sup.29).fwdarw.TQRQ). AS40 was randomly
chosen from the BC loop library described above. Both mutants were
expressed as soluble proteins in E. coli and were concentrated at
least to 1 mM, permitting NMR studies.
[0115] The mid-point of the thermal denaturation for both mutants
was approximately 69.degree. C., as compared to approximately
79.degree. C. for the wild-type protein. The results indicated that
the extensive mutations at the two surface loops did not
drastically decrease the stability of Fn3, and thus demonstrated
the feasibility of introducing a large number of mutations in both
loops.
[0116] Stability was also determined by guanidinium chloride
(GdnCl)- and urea-induced unfolding reactions. Preliminary
unfolding curves were recorded using a fluorometer equipped with a
motor-driven syringe; GdnCl or urea were added continuously to the
protein solution in the cuvette. Based on the preliminary unfolding
curves, separate samples containing varying concentration of a
denaturant were prepared and fluorescence (excitation at 290 nm,
emission at 300-400 nm) or CD (ellipticity at 222 and 215 nm) were
measured after the samples were equilibrated at the measurement
temperature for at least one hour. The curve was fitted by the
least-squares method to the equation for the two-state model
(Santoro & Bolen, 1988; Koide et al., 1993). The change in
protein concentration was compensated if required.
[0117] Once the reversibility of the thermal unfolding reaction is
established, the unfolding reaction is measured by a Microcal MC-2
differential scanning calorimeter (DSC). The cell (.about.1.3 ml)
will be filled with FnAb solution (0.1-1 mM) and .DELTA.Cp
(=.DELTA.H/.DELTA.T) will be recorded as the temperature is slowly
raised. T.sub.m (the midpoint of unfolding), .DELTA.H of unfolding
and .DELTA.G of unfolding is determined by fitting the transition
curve (Privalov & Potekhin, 1986) with the Origin software
provided by Microcal.
Thermal Unfolding
[0118] A temperature-induced unfolding experiment on Fn3 was
performed using circular dichroism (CD) spectroscopy to monitor
changes in secondary structure. The CD spectrum of the native Fn3
shows a weak signal near 222 nm (FIG. 3A), consistent with the
predominantly .beta.-structure of Fn3 (Perczel et al., 1992). A
cooperative unfolding transition is observed at 80-90.degree. C.,
clearly indicating high stability of Fn3 (FIG. 3B). The free energy
of unfolding could not be determined due to the lack of a
post-transition baseline. The result is consistent with the high
stability of the first Fn3 domain of human fibronectin (Litvinovich
et al., 1992), thus indicating that Fn3 domains are in general
highly stable.
Binding Assays
[0119] The binding reactions of monobodies were characterized
quantitatively using an isothermal titration calorimeter (ITC) and
fluorescence spectroscopy.
[0120] The enthalpy change (.DELTA.H) of binding were measured
using a Microcal Omega ITC (Wiseman et al., 1989). The sample cell
(.about.1.3 ml) was filled with Monobody solution (.ltoreq.100
changed according to K.sub.d), and the reference cell filled with
distilled water; the system was equilibrated at a given temperature
until a stable baseline is obtained; 5-20 .mu.l of ligand solution
(.ltoreq.2 mM) was injected by a motor-driven syringe within a
short duration (20 sec) followed by an equilibration delay (4
minutes); the injection was repeated and heat generation/absorption
for each injection was measured. From the change in the observed
heat change as a function of ligand concentration, .DELTA.H and
K.sub.d was determined (Wiseman et al., 1989). .DELTA.G and
.DELTA.S of the binding reaction was deduced from the two directly
measured parameters. Deviation from the theoretical curve was
examined to assess nonspecific (multiple-site) binding. Experiments
were also be performed by placing a ligand in the cell and
titrating with an FnAb. It should be emphasized that only ITC gives
direct measurement of .DELTA.H, thereby making it possible to
evaluate enthalpic and entropic contributions to the binding
energy. ITC was successfully used to monitor the binding reaction
of the D1.3 Ab (Tello et al., 1993; Bhat et al., 1994).
[0121] Intrinsic fluorescence is monitored to measure binding
reactions with K.sub.d in the sub-.mu.M range where the
determination of K.sub.d by ITC is difficult. Trp fluorescence
(excitation at .about.290 nm, emission at 300-350 nm) and Tyr
fluorescence (excitation at .about.260 nm, emission at .about.303
nm) is monitored as the Fn3-mutant solution (.ltoreq.10 .mu.M) is
titrated with ligand solution (.ltoreq.100 .mu.M). K.sub.d of the
reaction is determined by the nonlinear least-squares fitting of
the bimolecular binding equation. Presence of secondary binding
sites is examined using Scatchard analysis. In all binding assays,
control experiments are performed busing wild-type Fn3 (or
unrelated monobodies) in place of monobodies of interest.
II. Production of Fn3 Mutants with High Affinity and Specificity
Monobodies
[0122] Library screening was carried out in order to select
monobodies that bind to specific ligands. This is complementary to
the modeling approach described above. The advantage of
combinatorial screening is that one can easily produce and screen a
large number of variants (.gtoreq.10.sup.8), which is not feasible
with specific mutagenesis ("rational design") approaches. The phage
display technique (Smith, 1985; O'Neil & Hoess, 1995) was used
to effect the screening processes. Fn3 was fused to a phage coat
protein (pIII) and displayed on the surface of filamentous phages.
These phages harbor a single-stranded DNA genome that contains the
gene coding the Fn3 fusion protein. The amino acid sequence of
defined regions of Fn3 were randomized using a degenerate
nucleotide sequence, thereby constructing a library. Phages
displaying Fn3 mutants with desired binding capabilities were
selected in vitro, recovered and amplified. The amino acid sequence
of a selected clone can be identified readily by sequencing the Fn3
gene of the selected phage. The protocols of Smith (Smith &
Scott, 1993) were followed with minor modifications.
[0123] The objective was to produce Monobodies which have high
affinity to small protein ligands. HEL and the B1 domain of
staphylococcal protein G (hereafter referred to as protein G) were
used as ligands. Protein G is small (56 amino acids) and highly
stable (Minor & Kim, 1994; Smith et al., 1994). Its structure
was determined by NMR spectroscopy (Gronenborn et al., 1991) to be
a helix packed against a four-strand .beta.-sheet. The resulting
FnAb-protein G complexes (.about.150 residues) is one of the
smallest protein-protein complexes produced to date, well within
the range of direct NMR methods. The small size, the high stability
and solubility of both components and the ability to label each
with stable isotopes (.sup.13C and .sup.15N; see below for protein
G) make the complexes an ideal model system for NMR studies on
protein-protein interactions.
[0124] The successful loop replacement of Fn3 (the mutant D1.3-4)
demonstrate that at least ten residues can be mutated without the
loss of the global fold. Based on this, a library was first
constructed in which only residues in the FG loop are randomized.
After results of loop replacement experiments on the BC loop were
obtained, mutation sites were extended that include the BC loop and
other sites.
Construction of Fn3 Phage Display System
[0125] An M13 phage-based expression vector pASM1 has been
constructed as follows: an oligonucleotide coding the signal
peptide of OmpT was cloned at the 5' end of the Fn3 gene; a gene
fragment coding the C-terminal domain of M13 pIII was prepared from
the wild-type gene III gene of M13 mp18 using PCR (Corey et al.,
1993) and the fragment was inserted at the 3' end of the OmpT-Fn3
gene; a spacer sequence has been inserted between Fn3 and pIII. The
resultant fragment (OmpT-Fn3-pIII) was cloned in the multiple
cloning site of M13 mp18, where the fusion gene is under the
control of the lac promoter. This system will produce the Fn3-pIII
fusion protein as well as the wild-type pIII protein. The
co-expression of wild-type pIII is expected to reduce the number of
fusion pIII protein, thereby increasing the phage infectivity
(Corey et al., 1993) (five copies of pill are present on a phage
particle). In addition, a smaller number of fusion pIII protein may
be advantageous in selecting tight binding proteins, because the
chelating effect due to multiple binding sites should be smaller
than that with all five copies of fusion pIII (Bass et al., 1990).
This system has successfully displayed the serine protease trypsin
(Corey et al., 1993). Phages were produced and purified using E.
coli K91kan (Smith & Scott, 1993) according to a standard
method (Sambrook et al., 1989) except that phage particles were
purified by a second polyethylene glycol precipitation and acid
precipitation.
[0126] Successful display of Fn3 on fusion phages has been
confirmed by ELISA using an Ab against fibronectin (Sigma), clearly
indicating that it is feasible to construct libraries using this
system.
[0127] An alternative system using the fUSE5 (Parmley & Smith,
1988) may also be used. The Fn3 gene is inserted to fUSE5 using the
SfiI restriction sites introduced at the 5'- and 3'-ends of the Fn3
gene PCR. This system displays only the fusion pIII protein (up to
five copies) on the surface of a phage. Phages are produced and
purified as described (Smith & Scott, 1993). This system has
been used to display many proteins and is robust. The advantage of
fUSE5 is its low toxicity. This is due to the low copy number of
the replication form (RF) in the host, which in turn makes it
difficult to prepare a sufficient amount of RF for library
construction (Smith & Scott, 1993).
Construction of Libraries
[0128] The first library was constructed of the Fn3 domain
displayed on the surface of M13 phage in which seven residues
(77-83) in the FG loop (FIG. 4D) were randomized. Randomization
will be achieved by the use of an oligonucleotide containing
degenerated nucleotide sequence. A double-stranded nucleotide was
prepared by the same protocol as for gene synthesis (see above)
except that one strand had an (NNK).sub.6(NNG) sequence at the
mutation sites, where N corresponds to an equimolar mixture of A,
T, G and C and K corresponds to an equimolar mixture of G and T.
The (NNG) codon at residue 83 was required to conserve the Sad
restriction site (FIG. 2). The (NNK) codon codes all of the 20
amino acids, while the NNG codon codes 14. Therefore, this library
contained .about.10.sup.9 independent sequences. The library was
constructed by ligating the double-stranded nucleotide into the
wild-type phage vector, pASM1, and the transfecting E. coli XL1
blue (Stratagene) using electroporation. XL1 blue has the
lacI.sup.q phenotype and thus suppresses the expression of the
Fn3-pIII fusion protein in the absence of lac inducers. The initial
library was propagated in this way, to avoid selection against
toxic Fn3-pIII clones. Phages displaying the randomized Fn3-pIII
fusion protein were prepared by propagating phages with K91kan as
the host. K91kan does not suppress the production of the fusion
protein, because it does not have lacIq. Another library was also
generated in which the BC loop (residues 26-20) was randomized.
Selection of Displayed Monobodies
[0129] Screening of Fn3 phage libraries was performed using the
biopanning protocol (Smith & Scott, 1993); a ligand is
biotinylated and the strong biotin-streptavidin interaction was
used to immobilize the ligand on a streptavidin-coated dish.
Experiments were performed at room temperature (.about.22.degree.
C.). For the initial recovery of phages from a library, 10 .mu.g of
a biotinylated ligand were immobilized on a streptavidin-coated
polystyrene dish (35 mm, Falcon 1008) and then a phage solution
(containing .about.10'' pfu (plaque-forming unit)) was added. After
washing the dish with an appropriate buffer (typically TBST,
Tris-HCl (50 mM, pH 7.5), NaCl (150 mM) and Tween 20 (0.5%)), bound
phages were eluted by one or combinations of the following
conditions: low pH, an addition of a free ligand, urea (up to 6 M)
and, in the case of anti-protein G Monobodies, cleaving the protein
G-biotin linker by thrombin. Recovered phages were amplified using
the standard protocol using K91kan as the host (Sambrook et al.,
1989). The selection process were repeated 3-5 times to concentrate
positive clones. From the second round on, the amount of the ligand
were gradually decreased (to .about.1 .mu.g) and the biotinylated
ligand were mixed with a phage solution before transferring a dish
(G. P. Smith, personal communication). After the final round, 10-20
clones were picked, and their DNA sequence will be determined. The
ligand affinity of the clones were measured first by the
phage-ELISA method (see below).
[0130] To suppress potential binding of the Fn3 framework
(background binding) to a ligand, wild-type Fn3 may be added as a
competitor in the buffers. In addition, unrelated proteins (e.g.,
bovine serum albumin, cytochrome c and RNase A) may be used as
competitors to select highly specific Monobodies.
Binding Assay
[0131] The binding affinity of Monobodies on phage surface is
characterized semi-quantitatively using the phage ELISA technique
(Li et al., 1995). Wells of microtiter plates (Nunc) are coated
with a ligand protein (or with streptavidin followed by the binding
of a biotinylated ligand) and blocked with the Blotto solution
(Pierce). Purified phages (.about.10.sup.10 pfu) originating from
single plaques (M13)/colonies (fUSE5) are added to each well and
incubated overnight at 4.degree. C. After washing wells with an
appropriate buffer (see above), bound phages are detected by the
standard ELISA protocol using anti-M13 Ab (rabbit, Sigma) and
anti-rabbit Ig-peroxidase conjugate (Pierce) or using anti-M 13
Ab-peroxidase conjugate (Pharmacia). Colormetric assays are
performed using TMB (3,3',5,5'-tetramethylbenzidine, Pierce). The
high affinity of protein G to immunoglobulins present a special
problem; Abs cannot be used in detection. Therefore, to detect
anti-protein G Monobodies, fusion phages are immobilized in wells
and the binding is then measured using biotinylated protein G
followed by the detection using streptavidin-peroxidase
conjugate.
Production of Soluble Monobodies
[0132] After preliminary characterization of mutant Fn3s using
phage ELISA, mutant genes are subcloned into the expression vector
pEW1. Mutant proteins are produced as His.tag fusion proteins and
purified, and their conformation, stability and ligand affinity are
characterized.
III. Increased Stability of Fn3 Scaffolds
[0133] The definition of "higher stability" of a protein is the
ability of a protein to retain its three-dimensional structure
required for function at a higher temperature (in the case of
thermal denaturation), and in the presence of a higher
concentration of a denaturing chemical reagent such as guanidine
hydrochloride. This type of "stability" is generally called
"conformational stability." It has been shown that conformational
stability is correlated with resistance against proteolytic
degradation, i.e., breakdown of protein in the body (Kamtekar et
al. 1993).
[0134] Improving the conformational stability is a major goal in
protein engineering. Here, mutations have been developed by the
inventor that enhance the stability of the fibronectin type III
domain (Fn3). The inventor has developed a technology in which Fn3
is used as a scaffold to engineer artificial binding proteins
(Koide et al., 1998). It has been shown that many residues in the
surface loop regions of Fn3 can be mutated without disrupting the
overall structure of the Fn3 molecule, and that variants of Fn3
with a novel binding function can be engineered using combinatorial
library screening (Koide et al., 1998). The inventor found that,
although Fn3 is an excellent scaffold, Fn3 variants that contain
large number of mutations are destablized against chemical
denaturation, compared to the wild-type'Fn3 protein (Koide et al.,
1998). Thus, as the number of mutated positions are mutated in
order to engineer a new binding function, the stability of such Fn3
variants further decreases, ultimately leading to marginally stable
proteins. Because artificial binding proteins must maintain their
three-dimensional structure to be functional, stability limits the
number of mutations that can be introduced in the scaffold. Thus,
modifications of the Fn3 scaffold that increase its stability are
useful in that they allow one to introduce more mutations for
better function, and that they make it possible to use Fn3-based
engineered proteins in a wider range of applications.
[0135] The inventor found that wild-type Fn3 is more stable at
acidic pH than at neutral pH (Koide et al., 1998). The pH
dependence of Fn3 stability is characterized in FIG. 18. The pH
dependence curve has an apparent transition midpoint near pH 4
(FIG. 18). These results suggest that by identifying and removing
destablizing interactions in Fn3 one is able to improve the
stability of Fn3 at neutral pH. It should be noted that most
applications of engineered Fn3, such as diagnostics, therapeutics
and catalysts, are expected to be used near neutral pH, and thus it
is important to improve the stability at neutral pH. Studies by
other investigators have demonstrated that the optimization of
surface electrostatic properties can lead to a substantial increase
in protein stability (Peri et al. 2000, Spector et al. 1999,
Loladze et al. 1999, Grimsley et al. 1999).
[0136] The pH dependence of Fn3 stability suggests that amino acids
with pK.sub.a near 4 are involved in the observed transition. The
carboxyl groups of aspartic acid (Asp) and glutamic acid (Glu) have
pK.sub.a in this range (Creighton, T. E. 1993). It is well known
that if a carboxyl group has unfavorable (i.e. destabilizing)
interactions in a protein, its pK.sub.a is shifted to a higher
value from its standard, unperturbed value (Yang and Honig 1992).
Thus, the pK.sub.a values of all carboxyl groups in Fn3 were
determined using nuclear magnetic resonance (NMR) spectroscopy, to
identify carboxyl groups with unusual pK.sub.a's, as shown
below.
[0137] First, the .sup.13C resonance for the carboxyl carbon of
each Asp and Glu residue were assigned (FIG. 19). Next pH titration
of .sup.13C resonances was performed for these groups (FIG. 20).
The pK.sub.a values for these residues are listed in Table 3.
TABLE-US-00003 TABLE 3 pK.sub.a values for Asp and Glu residues in
Fn3. Residue pK.sub.a E9 5.09 E38 3.79 E47 3.94 D3 3.66 D7 3.54,
5.54* D23 3.54, 5.25* D67 4.18 D80 3.40 The standard deviation in
the pK.sub.a values are less than 0.05 pH units. *Data for D7 and
D23 were fitted with a transition curve with two pK.sub.a
values.
These results show that Asp 7 and 23, and Glu 9 have up-shifted
pK.sub.a's with respect to their unperturbed pK.sub.a's
(approximately 4.0), indicating that these residues are involved in
unfavorable interactions. In contrast, the other Asp and Glu
residues have pK.sub.a's close to the respective unperturbed
values, indicating that the carboxyl groups of these residues do
not significantly contribute to the stability of Fn3.
[0138] In the three-dimensional structure of Fn3 (Main et al.
1992), Asp 7 and 23, and Glu 9 form a patch on the surface (FIG.
21), with Asp 7 centrally located in the patch. This spatial
proximity of these negatively charged residues explains why these
residues have unfavorable interactions in Fn3. At low pH where
these residues are protonated and neutral, the unfavorable
interactions are expected to be mostly relieved. At the same time,
the structure suggests that the stability of Fn3 at neutral pH
could be improved if the electrostatic repulsion between these
three residues is removed. Because Asp 7 is centrally located among
the three residues, it was decided to mutate Asp 7. Two mutants
were prepared, D7N and D7K (i.e., the aspartic acid at amino acid
residue number 7 was substituted with an asparagine residue or a
lysine residue, respectively). The former replaces the negative
charge with a neutral residue of virtually the same size. The
latter places a positive charge at residue 7.
[0139] The degrees of stability of the mutant proteins were
characterized in thermal and chemical denaturation measurements. In
thermal denaturation measurements, denaturation of the Fn3 proteins
was monitored using circular dichroism spectroscopy at the
wavelength of 227 nm. All the proteins underwent a cooperative
transition (FIG. 22). From the transition curves, the midpoints of
the transition (T.sub.m) for the wild-type, D7N and D7K were
determined to be 62, 69 and 70.degree. C. in 0.02 M sodium
phosphate buffer (pH 7.0) containing 0.1 M sodium chloride and 6.2
M urea. Thus, the mutations increased the T.sub.m of wild-type Fn3
by 7-8.degree. C.
[0140] Chemical denaturation of Fn3 proteins was monitored using
fluorescence emission from the single Tip residue of Fn3 (FIG. 23).
The free energies of unfolding in the absence of guanidine HCl
(.DELTA.G.sup.0) were determined to be 7.4, 8.1 and 8.0 kcal/mol
for the wild-type, D7N and D7K, respectively (a larger
.DELTA.G.sup.0 indicates a higher stability). The two mutants were
again found to be more stable than the wild-type protein.
[0141] These results show that a point mutation on the surface can
significantly enhance the stability of Fn3. Because these mutations
are on the surface, they minimally alter the structure of Fn3, and
they can be easily introduced to other, engineered Fn3 proteins. In
addition, mutations at Glu 9 and/or Asp 23 also enhance the
stability of Fn3. Furthermore, mutations at one or more of these
three residues can be combined.
[0142] Thus, Fn3 is the fourth example of a monomeric
immunoglobulin-like scaffold that can be used for engineering
binding proteins. Successful selection of novel binding proteins
have also been based on minibody, tendamistat and "camelized"
immunoglobulin VH domain scaffolds (Martin et al., 1994; Davies
& Riechmann, 1995; McConnell & Hoess, 1995). The Fn3
scaffold has advantages over these systems. Bianchi et al. reported
that the stability of a minibody was 2.5 kcal/mol, significantly
lower than that of Ubi4-K. No detailed structural characterization
of minibodies has been reported to date. Tendamistat and the VH
domain contain disulfide bonds, and thus preparation of correctly
folded proteins may be difficult. Davies and Riechmann reported
that the yields of their camelized VH domains were less than 1 mg
per liter culture (Davies & Riechmann, 1996).
[0143] Thus, the Fn3 framework can be used as a scaffold for
molecular recognition. Its small size, stability and
well-characterized structure make Fn3 an attractive system. In
light of the ubiquitous presence of Fn3 in a wide variety of
natural proteins involved in ligand binding, one can engineer
Fn3-based binding proteins to different classes of targets.
[0144] The following examples are intended to illustrate but not
limit the invention.
Example I
Construction of the Fn3 Gene
[0145] A synthetic gene for tenth Fn3 of fibronectin (FIG. 1) was
designed on the basis of amino acid residue 1416-1509 of human
fibronectin (Kornblihtt, et al., 1985) and its three dimensional
structure (Main, et al., 1992). The gene was engineered to include
convenient restriction sites for mutagenesis and the so-called
"preferred codons" for high level protein expression (Gribskov, et
al., 1984) were used. In addition, a glutamine residue was inserted
after the N-terminal methionine in order to avoid partial
processing of the N-terminal methionine which often degrades NMR
spectra (Smith, et al., 1994). Chemical reagents were of the
analytical grade or better and purchased from Sigma Chemical
Company and J. T. Baker, unless otherwise noted. Recombinant DNA
procedures were performed as described in "Molecular Cloning"
(Sambrook, et al., 1989), unless otherwise stated. Custom
oligonucleotides were purchased from Operon Technologies.
Restriction and modification enzymes were from New England
Biolabs.
[0146] The gene was assembled in the following manner. First, the
gene sequence (FIG. 5) was divided into five parts with boundaries
at designed restriction sites: fragment 1, NdeI-PstI
(oligonucleotides FN1F and FN1R (Table 2); fragment 2, PstI-EcoRI
(FN2F and FN2R); fragment 3, EcoRI-SalI (FN3F and FN3R); fragment
4, SalI-SacI (FN4F and FN4R); fragment 5, SacI-BamHI (FN5F and
FN5R). Second, for each part, a pair of oligonucleotides which code
opposite strands and have complementary overlaps of approximately
15 bases was synthesized. These oligonucleotides were designated
FN1F-FN5R and are shown in Table 2. Third, each pair (e.g., FN1F
and FN1R) was annealed and single-strand regions were filled in
using the Klenow fragment of DNA polymerase. Fourth, the double
stranded oligonucleotide was digested with the relevant restriction
enzymes at the termini of the fragment and cloned into the
pBlueScript SK plasmid (Stratagene) which had been digested with
the same enzymes as those used for the fragments. The DNA sequence
of the inserted fragment was confirmed by DNA sequencing using an
Applied Biosystems DNA sequencer and the dideoxy termination
protocol provided by the manufacturer. Last, steps 2-4 were
repeated to obtain the entire gene.
[0147] The gene was also cloned into the pET3a and pET15b (Novagen)
vectors (pAS45 and pAS25, respectively). The maps of the plasmids
are shown in FIGS. 6 and 7. E. coli BL21 (DE3) (Novagen) containing
these vectors expressed the Fn3 gene under the control of
bacteriophage T7 promotor (Studier, et al., 1990); pAS24 expresses
the 96-residue Fn3 protein only, while pAS45 expresses Fn3 as a
fusion protein with poly-histidine peptide (His.tag). High level
expression of the Fn3 protein and its derivatives in E. coli was
detected as an intense band on SDS-PAGE stained with CBB.
[0148] The binding reaction of the monobodies is characterized
quantitatively by means of fluorescence spectroscopy using purified
soluble monobodies.
[0149] Intrinsic fluorescence is monitored to measure binding
reactions. Trp fluorescence (excitation at .about.290 nm, emission
at 300 350 nm) and Tyr fluorescence (excitation at .about.260 nm,
emission at .about.303 nm) is monitored as the Fn3-mutant solution
(.ltoreq.100 .mu.M) is titrated with a ligand solution. When a
ligand is fluorescent (e.g. fluorescein), fluorescence from the
ligand may be used. K.sub.d of the reaction will be determined by
the nonlinear least-squares fitting of the bimolecular binding
equation.
[0150] If intrinsic fluorescence cannot be used to monitor the
binding reaction, monobodies are labeled with fluorescein-NHS
(Pierce) and fluorescence polarization is used to monitor the
binding reaction (Burke et al., 1996).
Example II
Modifications to Include Restriction Sites in the Fn3 Gene
[0151] The restriction sites were incorporated in the synthetic Fn3
gene without changing the amino acid sequence Fn3. The positions of
the restriction sites were chosen so that the gene construction
could be completed without synthesizing long (>60 bases)
oligonucleotides and so that two loop regions could be mutated
(including by randomization) by the cassette mutagenesis method
(i.e., swapping a fragment with another synthetic fragment
containing mutations). In addition, the restriction sites were
chosen so that most sites were unique in the vector for phage
display. Unique restriction sites allow one to recombine monobody
clones which have been already selected in order to supply a larger
sequence space.
Example III
Construction of M13 Phage Display Libraries
[0152] A vector for phage display, pAS38 (for its map, see FIG. 8)
was constructed as follows. The XbaI-BamHI fragment of pET12a
encoding the signal peptide of OmpT was cloned at the 5' end of the
Fn3 gene. The C-terminal region (from the FN5F and FN5R
oligonucleotides, see Table 2) of the Fn3 gene was replaced with a
new fragment consisting of the FN5F and FN5R' oligonucleotides
(Table 2) which introduced a MluI site and a linker sequence for
making a fusion protein with the pIII protein of bacteriophage M13.
A gene fragment coding the C-terminal domain of M13 pIII was
prepared from the wild-type gene III of M13 mp18 using PCR (Corey,
et al., 1993) and the fragment was inserted at the 3' end of the
OmpT-Fn3 fusion gene using the MluI and HindIII sites.
[0153] Phages were produced and purified using a helper phage,
M13K07, according to a standard method (Sambrook, et al., 1989)
except that phage particles were purified by a second polyethylene
glycol precipitation. Successful display of Fn3 on fusion phages
was confirmed by ELISA (Harlow & Lane, 1988) using an antibody
against fibronectin (Sigma) and a custom anti-FN3 antibody
(Cocalico Biologicals, PA, USA).
Example IV
Libraries Containing Loop Variegations in the AB Loop
[0154] A nucleic acid phage display library having variegation in
the AB loop is prepared by the following methods. Randomization is
achieved by the use of oligonucleotides containing degenerated
nucleotide sequence. Residues to be variegated are identified by
examining the X-ray and NMR structures of Fn3 (Protein Data Bank
accession numbers, 1FNA and 1TTF, respectively). Oligonucleotides
containing NNK (N and K here denote an equimolar mixture of A, T,
G, and C and an equimolar mixture of G and T, respectively) for the
variegated residues are synthesized (see oligonucleotides BC3, FG2,
FG3, and FG4 in Table 2 for example). The NNK mixture codes for all
twenty amino acids and one termination codon (TAG). TAG, however,
is suppressed in the E. coli XL-1 blue. Single-stranded DNAs of
pAS38 (and its derivatives) are prepared using a standard protocol
(Sambrook, et al., 1989).
[0155] Site-directed mutagenesis is performed following published
methods (see for example, Kunkel, 1985) using a Muta-Gene kit
(BioRad). The libraries are constructed by electroporation of E.
coli XL-1 Blue electroporation competent cells (200 .mu.l;
Stratagene) with 1 .mu.g of the plasmid DNA using a BTX electrocell
manipulator ECM 395 1 mm gap cuvette. A portion of the transformed
cells is plated on an LB-agar plate containing ampicillin (100
.mu.g/ml) to determine the transformation efficiency. Typically,
3.times.10 transformants are obtained with 1 .mu.g of DNA, and thus
a library contains 10.sup.8 to 10.sup.9 independent clones.
Phagemid particles were prepared as described above.
Example V
Loop Variegations in the BC, CD, DE, EF or FG Loop
[0156] A nucleic acid phage display library having five variegated
residues (residues number 26-30) in the BC loop, and one having
seven variegated residues (residue numbers 78-84) in the FG loop,
was prepared using the methods described in Example IV above. Other
nucleic acid phage display libraries having variegation in the CD,
DE or EF loop can be prepared by similar methods.
Example VI
Loop Variegations in the FG and BC Loop
[0157] A nucleic acid phage display library having seven variegated
residues (residues number 78-84) in the FG loop and five variegated
residues (residue number 26-30) in the BC loop was prepared.
Variegations in the BC loop were prepared by site-directed
mutagenesis (Kunkel, et al.) using the BC3 oligonucleotide
described in Table 1. Variegations in the FG loop were introduced
using site-directed mutagenesis using the BC loop library as the
starting material, thereby resulting in libraries containing
variegations in both BC and FG loops. The oligonucleotide FG2 has
variegating residues 78-84 and oligonucleotide FG4 has variegating
residues 77-81 and a deletion of residues 82-84.
[0158] A nucleic acid phage display library having five variegated
residues (residues 78-84) in the FG loop and a three residue
deletion (residues 82-84) in the FG loop, and five variegated
residues (residues 26-30) in the BC loop, was prepared. The shorter
FG loop was made in an attempt to reduce the flexibility of the FG
loop; the loop was shown to be highly flexible in Fn3 by the NMR
studies of Main, et al. (1992). A highly flexible loop may be
disadvantageous to forming a binding site with a high affinity (a
large entropy loss is expected upon the ligand binding, because the
flexible loop should become more rigid). In addition, other Fn3
domains (besides human) have shorter FG loops (for sequence
alignment, see FIG. 12 in Dickinson, et al. (1994)).
[0159] Randomization was achieved by the use of oligonucleotides
containing degenerate nucleotide sequence (oligonucleotide BC3 for
variegating the BC loop and oligonucleotides FG2 and FG4 for
variegating the FG loops).
[0160] Site-directed mutagenesis was performed following published
methods (see for example, Kunkel, 1985). The libraries were
constructed by electrotransforming E. coli XL-1 Blue (Stratagene).
Typically a library contains 10.sup.8 to 10.sup.9 independent
clones. Library 2 contains five variegated residues in the BC loop
and seven variegated residues in the FG loop. Library 4 contains
five variegated residues in each of the BC and FG loops, and the
length of the FG loop was shortened by three residues.
Example VII
fd Phage Display Libraries Constructed with Loop Variegations
[0161] Phage display libraries are constructed using the fd phage
as the genetic vector. The Fn3 gene is inserted in fUSE5 (Parmley
& Smith, 1988) using SfiI restriction sites which are
introduced at the 5' and 3' ends of the Fn3 gene using PCR. The
expression of this phage results in the display of the fusion pIII
protein on the surface of the fd phage. Variegations in the Fn3
loops are introduced using site-directed mutagenesis as described
hereinabove, or by subcloning the Fn3 libraries constructed in M13
phage into the fUSE5 vector.
Example VIII
Other Phage Display Libraries
[0162] T7 phage libraries (Novagen, Madison, Wis.) and bacterial
pili expression systems (Invitrogen) are also useful to express the
Fn3 gene.
Example IX
Isolation of Polypeptides which Bind to Macromolecular
Structures
[0163] The selection of phage-displayed monobodies was performed
following the protocols of Barbas and coworkers (Rosenblum &
Barbas, 1995). Briefly, approximately 1 .mu.g of a target molecule
("antigen") in sodium carbonate buffer (100 mM, pH 8.5) was
immobilized in the wells of a microtiter plate (Maxisorp, Nunc) by
incubating overnight at 4.degree. C. in an air tight container.
After the removal of this solution, the wells were then blocked
with a 3% solution of BSA (Sigma, Fraction V) in TBS by incubating
the plate at 37.degree. C. for 1 hour. A phagemid library solution
(50 .mu.l) containing approximately 10.sup.12 colony forming units
(cfu) of phagemid was absorbed in each well at 37.degree. C. for 1
hour. The wells were then washed with an appropriate buffer
(typically TBST, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5%
Tween20) three times (once for the first round). Bound phage were
eluted by an acidic solution (typically, 0.1 M glycine-HCl, pH 2.2;
50 .mu.l) and recovered phage were immediately neutralized with 3
.mu.l of Tris solution. Alternatively, bound phage were eluted by
incubating the wells with 50 .mu.l of TBS containing the antigen
(1-10 .mu.M). Recovered phage were amplified using the standard
protocol employing the XL1Blue cells as the host (Sambrook, et
al.). The selection process was repeated 5-6 times to concentrate
positive clones. After the final round, individual clones were
picked and their binding affinities and DNA sequences were
determined.
[0164] The binding affinities of monobodies on the phage surface
were characterized using the phage ELISA technique (Li, et al.,
1995). Wells of microtiter plates (Nunc) were coated with an
antigen and blocked with BSA. Purified phages (10.sup.8-10.sup.11
cfu) originating from a single colony were added to each well and
incubated 2 hours at 37.degree. C. After washing wells with an
appropriate buffer (see above), bound phage were detected by the
standard ELISA protocol using anti-M13 antibody (rabbit, Sigma) and
anti-rabbit Ig-peroxidase conjugate (Pierce). Colorimetric assays
were performed using Turbo-TMB (3,3',5,5'-tetramethylbenzidine,
Pierce) as a substrate.
[0165] The binding affinities of monobodies on the phage surface
were further characterized using the competition ELISA method
(Djavadi-Ohaniance, et al., 1996). In this experiment, phage ELISA
is performed in the same manner as described above, except that the
phage solution contains a ligand at varied concentrations. The
phage solution was incubated a 4.degree. C. for one hour prior to
the binding of an immobilized ligand in a microtiter plate well.
The affinities of phage displayed monobodies are estimated by the
decrease in ELISA signal as the free ligand concentration is
increased.
[0166] After preliminary characterization of monobodies displayed
on the surface of phage using phage ELISA, genes for positive
clones were subcloned into the expression vector pAS45. E. coli
BL21(DE3) (Novagen) was transformed with an expression vector
(pAS45 and its derivatives). Cells were grown in M9 minimal medium
and M9 medium supplemented with Bactotryptone (Difco) containing
ampicillin (200 .mu.g/ml). For isotopic labeling, .sup.15N
NH.sub.4Cl and/or .sup.13C glucose replaced unlabeled components.
Stable isotopes were purchased from Isotec and Cambridge Isotope
Labs. 500 ml medium in a 2 1 baffle flask was inoculated with 10 ml
of overnight culture and agitated at approximately 140 rpm at
37.degree. C. IPTG was added at a final concentration of 1 mM to
induce protein expression when OD(600 nm) reached approximately
1.0. The cells were harvested by centrifugation 3 hours after the
addition of IPTG and kept frozen at -70.degree. C. until used.
[0167] Fn3 and monobodies with His.tag were purified as follows.
Cells were suspended in 5 ml/(g cell) of 50 mM Tris (pH 7.6)
containing 1 mM phenylmethylsulfonyl fluoride. HEL (Sigma, 3.times.
crystallized) was added to a final concentration of 0.5 mg/ml.
After incubating the solution for 30 min at 37.degree. C., it was
sonicated so as to cause cell breakage three times for 30 seconds
on ice. Cell debris was removed by centrifugation at 15,000 rpm in
an Sorval RC-2B centrifuge using an SS-34 rotor. Concentrated
sodium chloride is added to the solution to a final concentration
of 0.5 M. The solution was then applied to a 1 ml HisTrap.TM.
chelating column (Pharmacia) preloaded with nickel chloride (0.1 M,
1 ml) and equilibrated in the Tris buffer (50 mM, pH 8.0)
containing 0.5 M sodium chloride. After washing the column with the
buffer, the bound protein was eluted with a Tris buffer (50 mM, pH
8.0) containing 0.5 M imidazole. The His.tag portion was cleaved
off, when required, by treating the fusion protein with thrombin
using the protocol supplied by Novagen (Madison, Wis.). Fn3 was
separated from the His.tag peptide and thrombin by a
Resources.RTM.column (Pharmacia) using a linear gradient of sodium
chloride (0-0.5 M) in sodium acetate buffer (20 mM, pH 5.0).
[0168] Small amounts of soluble monobodies were prepared as
follows. XL-1 Blue cells containing pAS38 derivatives (plasmids
coding Fn3-pIII fusion proteins) were grown in LB media at
37.degree. C. with vigorous shaking until OD(600 nm) reached
approximately 1.0; IPTG was added to the culture to a final
concentration of 1 mM, and the cells were further grown overnight
at 37.degree. C. Cells were removed from the medium by
centrifugation, and the supernatant was applied to a microtiter
well coated with a ligand. Although XL-1 Blue cells containing
pAS38 and its derivatives express FN3-pIII fusion proteins, soluble
proteins are also produced due to the cleavage of the linker
between the Fn3 and pIII regions by proteolytic activities of E.
coli (Rosenblum & Barbas, 1995). Binding of a monobody to the
ligand was examined by the standard ELISA protocol using a custom
antibody against Fn3 (purchased from Cocalico Biologicals,
Reamstown, Pa.). Soluble monobodies obtained from the periplasmic
fraction of E. coli cells using a standard osmotic shock method
were also used.
Example X
Ubiquitin Binding Monobody
[0169] Ubiquitin is a small (76 residue) protein involved in the
degradation pathway in eukaryotes. It is a single domain globular
protein. Yeast ubiquitin was purchased from Sigma Chemical Company
and was used without further purification.
[0170] Libraries 2 and 4, described in Example VI above, were used
to select ubiquitin-binding monobodies. Ubiquitin (1 .mu.g in 50
.mu.l sodium bicarbonate buffer (100 mM, pH 8.5)) was immobilized
in the wells of a microtiter plate, followed by blocking with BSA
(3% in TBS). Panning was performed as described above. In the first
two rounds, 1 .mu.g of ubiquitin was immobilized per well, and
bound phage were elute with an acidic solution. From the third to
the sixth rounds, 0.1 .mu.g of ubiquitin was immobilized per well
and the phage were eluted either with an acidic solution or with
TBS containing 10 .mu.M ubiquitin.
[0171] Binding of selected clones was tested first in the
polyclonal mode, i.e., before isolating individual clones. Selected
clones from all libraries showed significant binding to ubiquitin.
These results are shown in FIG. 9. The binding to the immobilized
ubiquitin of the clones was inhibited almost completely by less
than 30 .mu.M soluble ubiquitin in the competition ELISA
experiments (see FIG. 10). The sequences of the BC and FG loops of
ubiquitin-binding monobodies is shown in Table 4.
TABLE-US-00004 TABLE 4 Sequences of ubiquitin-binding monobodies
Occurrence (if more Name BC loop FG loop than one) 211 CARRA
RWIPLAK 2 (SEQ ID NO: 31) (SEQ ID NO: 32) 212 CWRRA RWVGLAW (SEQ ID
NO: 33) (SEQ ID NO: 34) 213 CKHRR FADLWWR (SEQ ID NO: 35) (SEQ ID
NO: 36) 214 CRRGR RGFMWLS (SEQ ID NO: 37) (SEQ ID NO: 38) 215 CNWRR
RAYRYRW (SEQ ID NO: 39) (SEQ ID NO: 40) 411 SRLRR PPWRV 9 (SEQ ID
NO: 41) (SEQ ID NO: 42) 422 ARWTL RRWWW (SEQ ID NO: 43) (SEQ ID NO:
44) 424 GQRTF RRWWA (SEQ ID NO: 45) (SEQ ID NO: 46)
[0172] The 411 clone, which was the most enriched clone, was
characterized using phage ELISA. The 411 clone showed selective
binding and inhibition of binding in the presence of about 10 .mu.M
ubiquitin in solution (FIG. 11).
Example XI
Methods for the Immobilization of Small Molecules
[0173] Target molecules were immobilized in wells of a microtiter
plate (Maxisorp, Nunc) as described hereinbelow, and the wells were
blocked with BSA. In addition to the use of carrier protein as
described below, a conjugate of a target molecule in biotin can be
made. The biotinylated ligand can then be immobilized to a
microtiter plate well which has been coated with streptavidin.
[0174] In addition to the use of a carrier protein as described
below, one could make a conjugate of a target molecule and biotin
(Pierce) and immobilize a biotinylated ligand to a microtiter plate
well which has been coated with streptavidin (Smith and Scott,
1993).
[0175] Small molecules may be conjugated with a carrier protein
such as bovine serum albumin (BSA, Sigma), and passively adsorbed
to the microtiter plate well. Alternatively, methods of chemical
conjugation can also be used. In addition, solid supports other
than microtiter plates can readily be employed.
Example XII
Fluorescein Binding Monobody
[0176] Fluorescein has been used as a target for the selection of
antibodies from combinatorial libraries (Barbas, et al. 1992).
NHS-fluorescein was obtained from Pierce and used according to the
manufacturer's instructions in preparing conjugates with BSA
(Sigma). Two types of fluorescein-BSA conjugates were prepared with
approximate molar ratios of 17 (fluorescein) to one (BSA).
[0177] The selection process was repeated 5-6 times to concentrate
positive clones. In this experiment, the phage library was
incubated with a protein mixture (BSA, cytochrome C (Sigma, Horse)
and RNaseA (Sigma, Bovine), 1 mg/ml each) at room temperature for
30 minutes, prior to the addition to ligand coated wells. Bound
phage were eluted in TBS containing 10 .mu.M soluble fluorescein,
instead of acid elution. After the final round, individual clones
were picked and their binding affinities (see below) and DNA
sequences were determined.
TABLE-US-00005 TABLE 5 BC FG Clones from Library #2 WT AVTVR
RGDSPAS (SEQ ID NO: 47) (SEQ ID NO: 48) pLB24.1 CNWRR RAYRYRW (SEQ
ID NO: 49) (SEQ ID NO: 50) pLB24.2 CMWRA RWGMLRR (SEQ ID NO: 51)
(SEQ ID NO: 52) pLB24.3 ARMRE RWLRGRY (SEQ ID NO: 53) (SEQ ID NO:
54) pLB24.4 CARRR RRAGWGW (SEQ ID NO: 55) (SEQ ID NO: 56) pLB24.5
CNWRR RAYRYRW (SEQ ID NO: 57) (SEQ ID NO: 58) pLB24.6 RWRER RHPWTER
(SEQ ID NO: 59) (SEQ ID NO: 60) pLB24.7 CNWRR RAYRYRW (SEQ ID NO:
61) (SEQ ID NO: 62) pLB24.8 ERRVP RLLLWQR (SEQ ID NO: 63) (SEQ ID
NO: 64) pLB24.9 GRGAG FGSFERR (SEQ ID NO: 65) (SEQ ID NO: 66)
pLB24.11 CRWTR RRWFDGA (SEQ ID NO: 67) (SEQ ID NO: 68) pLB24.12
CNWRR RAYRYRW (SEQ ID NO: 69) (SEQ ID NO: 70) Clones from Library
#4 WT AVTVR GRGDS (SEQ ID NO: 71) (SEQ ID NO: 72) pLB25.1 GQRTF
RRWWA (SEQ ID NO: 73) (SEQ ID NO: 74) pLB25.2 GQRTF RRWWA (SEQ ID
NO: 75) (SEQ ID NO: 76) pLB25.3 GQRTF RRWWA (SEQ ID NO: 77) (SEQ ID
NO: 78) pLB25.4 LRYRS GWRWR (SEQ ID NO: 79) (SEQ ID NO: 80) pLB25.5
GQRTF RRWWA (SEQ ID NO: 81) (SEQ ID NO: 82) pLB25.6 GQRTF RRWWA
(SEQ ID NO: 83) (SEQ ID NO: 84) pLB25.7 LRYRS GWRWR (SEQ ID NO: 85)
(SEQ ID NO: 86) pLB25.9 LRYRS GWRWR (SEQ ID NO: 87) (SEQ ID NO: 88)
pLB25.11 GQRTF RRWWA (SEQ ID NO: 89) (SEQ ID NO: 90) pLB25.12 LRYRS
GWRWR (SEQ ID NO: 91) (SEQ ID NO: 92)
[0178] Preliminary characterization of the binding affinities of
selected clones were performed using phage ELISA and competition
phage ELISA (see FIG. 12 (Fluorescein-1) and FIG. 13
(Fluorescein-2)). The four clones tested showed specific binding to
the ligand-coated wells, and the binding reactions are inhibited by
soluble fluorescein (see FIG. 13).
Example XIII
Digoxigenin Binding Monobody
[0179] Digoxigenin-3-O-methyl-carbonyl-e-aminocapronic acid-NHS
(Boehringer Mannheim) is used to prepare a digoxigenin-BSA
conjugate. The coupling reaction is performed following the
manufacturers' instructions. The digoxigenin-BSA conjugate is
immobilized in the wells of a microtiter plate and used for
panning. Panning is repeated 5 to 6 times to enrich binding clones.
Because digoxigenin is sparingly soluble in aqueous solution, bound
phages are eluted from the well using acidic solution. See Example
XIV.
Example XIV
TSAC (Transition State Analog Compound) Binding Monobodies
[0180] Carbonate hydrolyzing monobodies are selected as follows. A
transition state analog for carbonate hydrolysis, 4-nitrophenyl
phosphonate is synthesized by an Arbuzov reaction as described
previously (Jacobs and Schultz, 1987). The phosphonate is then
coupled to the carrier protein, BSA, using carbodiimide, followed
by exhaustive dialysis (Jacobs and Schultz, 1987). The hapten-BSA
conjugate is immobilized in the wells of a microtiter plate and
monobody selection is performed as described above. Catalytic
activities of selected monobodies are tested using 4-nitrophenyl
carbonate as the substrate.
[0181] Other haptens useful to produce catalytic monobodies are
summarized in H. Suzuki (1994) and in N. R. Thomas (1994).
Example XV
NMR Characterization of Fn3 and Comparison of the Fn3 Secreted by
Yeast with that Secreted by E. coli
[0182] Nuclear magnetic resonance (NMR) experiments are performed
to identify the contact surface between FnAb and a target molecule,
e.g., monobodies to fluorescein, ubiquitin, RNaseA and soluble
derivatives of digoxigenin. The information is then be used to
improve the affinity and specificity of the monobody. Purified
monobody samples are dissolved in an appropriate buffer for NMR
spectroscopy using Amicon ultrafiltration cell with a YM-3
membrane. Buffers are made with 90% H.sub.2O/10% D.sub.2O
(distilled grade, Isotec) or with 100% D.sub.2O. Deuterated
compounds (e.g. acetate) are used to eliminate strong signals from
them.
[0183] NMR experiments are performed on a Varian Unity INOVA 600
spectrometer equipped with four RF channels and a triple resonance
probe with pulsed field gradient capability. NMR spectra are
analyzed using processing programs such as Felix (Molecular
Simulations), nmrPipe, PIPP, and CAPP (Garrett, et al., 1991;
Delaglio, et al., 1995) on UNIX workstations. Sequence specific
resonance assignments are made using well-established strategy
using a set of triple resonance experiments (CBCA(CO)NH and HNCACB)
(Grzesiek & Bax, 1992; Wittenkind & Mueller, 1993).
[0184] Nuclear Overhauser effect (NOE) is observed between .sup.1H
nuclei closer than approximately 5 .ANG., which allows one to
obtain information on interproton distances. A series of double-
and triple-resonance experiments (Table 6; for recent reviews on
these techniques, see Bax & Grzesiek, 1993 and Kay, 1995) are
performed to collect distance (i.e. NOE) and dihedral angle
(J-coupling) constraints. Isotope-filtered experiments are
performed to determine resonance assignments of the bound ligand
and to obtain distance constraints within the ligand and those
between FnAb and the ligand. Details of sequence specific resonance
assignments and NOE peak assignments have been described in detail
elsewhere (Clore & Gronenborn, 1991; Pascal, et al., 1994b;
Metzler, et al., 1996).
TABLE-US-00006 TABLE 6 NMR experiments for structure
characterization Experiment Name Reference 1. reference spectra
2D-.sup.1H, .sup.15N-HSQC (Bodenhausen & Ruben, 1980; Kay, et
al., 1992) 2D-.sup.1H, .sup.13C-HSQC (Bodenhausen & Ruben,
1980; Vuister & Bax, 1992) 2. backbone and side chain resonance
assignments of .sup.13C/.sup.15N- labeled protein 3D-CBCA(CO)NH
(Grzesiek & Bax, 1992) 3D-HNCACB (Wittenkind & Mueller,
1993) 3D-C(CO)NH (Logan et al., 1992; Grzesiek et al., 1993)
3D-H(CCO)NH 3D-HBHA(CBCACO)NH (Grzesiek & Bax, 1993)
3D-HCCH-TOCSY (Kay et al., 1993) 3D-HCCH-COSY (Ikura et al., 1991)
3D-.sup.1H, .sup.15N-TOCSY-HSQC (Zhang et al., 1994)
2D-HB(CBCDCE)HE (Yamazaki et al., 1993) 3. resonance assignments of
unlabeled ligand 2D-isotope-filtered .sup.1H-TOCSY
2D-isotope-filtered .sup.1H-COSY 2D-isotope-filtered .sup.1H-NOESY
(Ikura & Bax, 1992) 4. structural constraints within labeled
protein 3D-.sup.1H, .sup.15N-NOESY-HSQC (Zhang et al., 1994)
4D-.sup.1H, .sup.13C-HMQC-NOESY-HMQC (Vuister et al., 1993)
4D-.sup.1H, .sup.13C, .sup.15N-HSQC-NOESY-HSQC (Muhandiram et al.,
within unlabeled ligand 1993; Pascal et al., 1994a)
2D-isotope-filtered .sup.1H-NOESY (Ikura & Bax, 1992)
interactions between protein and ligand 3D-isotope-filtered
.sup.1H, .sup.15N-NOESY- HSQC 3D-isotope-filtered .sup.1H,
.sup.13C-NOESY- (Lee et al., 1994) HSQC 5. dihedral angle
constraints J-molulated .sup.1H, .sup.15N-HSQC (Billeter et al.,
1992) 3D-HNHB (Archer et al., 1991)
[0185] Backbone .sup.1H, .sup.15N and .sup.13C resonance
assignments for a monobody are compared to those for wild-type Fn3
to assess structural changes in the mutant. Once these data
establish that the mutant retains the global structure, structural
refinement is performed using experimental NOE data. Because the
structural difference of a monobody is expected to be minor, the
wild-type structure can be used as the initial model after
modifying the amino acid sequence. The mutations are introduced to
the wild-type structure by interactive molecular modeling, and then
the structure is energy-minimized using a molecular modeling
program such as Quanta (Molecular Simulations). Solution structure
is refined using cycles of dynamical simulated annealing (Nilges et
al., 1988) in the program X-PLOR (Brunger, 1992). Typically, an
ensemble of fifty structures is calculated. The validity of the
refined structures is confirmed by calculating a fewer number of
structures from randomly generated initial structures in X-PLOR
using the YASAP protocol (Nilges, et al., 1991). Structure of a
monobody-ligand complex is calculated by first refining both
components individually using intramolecular NOEs, and then docking
the two using intermolecular NOEs.
[0186] For example, the .sup.1H, .sup.15N-HSQC spectrum for the
fluorescein-binding monobody LB25.5 is shown in FIG. 14. The
spectrum shows a good dispersion (peaks are spread out) indicating
that LB25.5 is folded into a globular conformation. Further, the
spectrum resembles that for the wild-type Fn3, showing that the
overall structure of LB25.5 is similar to that of Fn3. These
results demonstrate that ligand-binding monobodies can be obtained
without changing the global fold of the Fn3 scaffold.
[0187] Chemical shift perturbation experiments are performed by
forming the complex between an isotope-labeled FnAb and an
unlabeled ligand. The formation of a stoichiometric complex is
followed by recording the HSQC spectrum. Because chemical shift is
extremely sensitive to nuclear environment, formation of a complex
usually results in substantial chemical shift changes for
resonances of amino acid residues in the interface. Isotope-edited
NMR experiments (2D HSQC and 3D CBCA(CO)NH) are used to identify
the resonances that are perturbed in the labeled component of the
complex; i.e. the monobody. Although the possibility of artifacts
due to long-range conformational changes must always be considered,
substantial differences for residues clustered on continuous
surfaces are most likely to arise from direct contacts (Chen et
al., 1993; Gronenborn & Clore, 1993).
[0188] An alternative method for mapping the interaction surface
utilizes amide hydrogen exchange (HX) measurements. HX rates for
each amide proton are measured for .sup.15N labeled monobody both,
free and complexed with a ligand. Ligand binding is expected to
result in decreased amide HX rates for monobody residues in the
interface between the two proteins, thus identifying the binding
surface. HX rates for monobodies in the complex are measured by
allowing HX to occur for a variable time following transfer of the
complex to D.sub.2O; the complex is dissociated by lowering pH and
the HSQC spectrum is recorded at low pH where amide HX is slow. Fn3
is stable and soluble at low pH, satisfying the prerequisite for
the experiments.
Example XVI
Construction and Analysis of Fn3-Display System Specific for
Ubiquitin
[0189] An Fn3-display system was designed and synthesized,
ubiquitin-binding clones were isolated and a major Fn3 mutant in
these clones was biophysically characterized.
[0190] Gene construction and phage display of Fn3 was performed as
in Examples I and II above. The Fn3-phage pIII fusion protein was
expressed from a phagemid-display vector, while the other
components of the M13 phage, including the wild-type pIII, were
produced using a helper phage (Bass et al., 1990). Thus, a phage
produced by this system should contain less than one copy of Fn3
displayed on the surface. The surface display of Fn3 on the phage
was detected by ELISA using an anti-Fn3 antibody. Only phages
containing the Fn3-pIII fusion vector reacted with the
antibody.
[0191] After confirming the phage surface to display Fn3, a phage
display library of Fn3 was constructed as in Example III. Random
sequences were introduced in the BC and FG loops. In the first
library, five residues (77-81) were randomized and three residues
(S2-84) were deleted from the FG loop. The deletion was intended to
reduce the flexibility and improve the binding affinity of the FG
loop. Five residues (26-30) were also randomized in the BC loop in
order to provide a larger contact surface with the target molecule.
Thus, the resulting library contains five randomized residues in
each of the BC and FG loops (Table 7). This library contained
approximately 10.sup.8 independent clones.
Library Screening
[0192] Library screening was performed using ubiquitin as the
target molecule. In each round of panning, Fn3-phages were absorbed
to a ubiquitin-coated surface, and bound phages were eluted
competitively with soluble ubiquitin. The recovery ratio improved
from 4.3.times.10''.sup.7 in the second round to
4.5.times.10.sup.-6 in the fifth round, suggesting an enrichment of
binding clones. After five founds of panning, the amino acid
sequences of individual clones were determined (Table 7).
TABLE-US-00007 TABLE 7 Sequences in the variegated loops of
enriched clones Name BC loop FG loop Frequency Wild GCAGTTACCGTGCGT
GGCCGTGGTGACAGCCCAGCGAGC -- Type (SEQ ID NO: 93) (SEQ ID NO: 95)
AlaValThrValArg GlyArgGlyAspSerProAlaSer (SEQ ID NO: 94) (SEQ ID
NO: 96) Library.sup.a NNKNNKNNKNNKNNK NNKNNKNNKNNKNNK--------- -- X
X X X X X X X X X (deletion) clone1 TCGAGGTTGCGGCGG CCGCCGTGGAGGGTG
9 (Ubi4) (SEQ ID NO: 97) (SEQ ID NO: 99) SerArgLeuArgArg
ProProTrpArgVal (SEQ ID NO: 98) (SEQ ID NO: 100) clone2
GGTCAGCGAACTTTT AGGCGGTGGTGGGCT 1 (SEQ ID NO: 101) (SEQ ID NO: 103)
GlyGlnArgThrPhe ArgArgTrpTrpAla (SEQ ID NO: 102) (SEQ ID NO: 104)
clone3 GCGAGGTGGACGCTT AGGCGGTGGTGGTGG 1 (SEQ ID NO: 105) (SEQ ID
NO: 107) AlaArgTrpThrLeu ArgArgTrpTrpTrp (SEQ ID NO: 106) (SEQ ID
NO: 108) .sup.aN denotes an equimolar mixture of A, T, G and C; K
denotes an equimolar mixture of G and T.
[0193] A clone, dubbed Ubi4, dominated the enriched pool of Fn3
variants. Therefore, further investigation was focused on this Ubi4
clone. Ubi4 contains four mutations in the BC loop (Arg 30 in the
BC loop was conserved) and five mutations and three deletions in
the FG loop. Thus 13% (12 out of 94) of the residues were altered
in Ubi4 from the wild-type sequence.
[0194] FIG. 15 shows a phage ELISA analysis of Ubi4. The Ubi4 phage
binds to the target molecule, ubiquitin, with a significant
affinity, while a phage displaying the wild-type Fn3 domain or a
phase with no displayed molecules show little detectable binding to
ubiquitin (FIG. 15a). In addition, the Ubi4 phage showed a somewhat
elevated level of background binding to the control surface lacking
the ubiquitin coating. A competition ELISA experiments shows the
IC.sub.50 (concentration of the free ligand which causes 50%
inhibition of binding) of the binding reaction is approximately 5
(FIG. 15b). BSA, bovine ribonuclease A and cytochrome C show little
inhibition of the Ubi4-ubiquitin binding reaction (FIG. 15c),
indicating that the binding reaction of Ubi4 to ubiquitin does
result from specific binding.
Characterization of a Mutant Fn3 Protein
[0195] The expression system yielded 50-100 mg Fn3 protein per
liter culture. A similar level of protein expression was observed
for the Ubi4 clone and other mutant Fn3 proteins.
[0196] Ubi4-Fn3 was expressed as an independent protein. Though a
majority of Ubi4 was expressed in E. coli as a soluble protein, its
solubility was found to be significantly reduced as compared to
that of wild-type Fn3. Ubi4 was soluble up to .about.20 .mu.M at
low pH, with much lower solubility at neutral pH. This solubility
was not high enough for detailed structural characterization
using
NMR Spectroscopy or X-Ray Crystallography.
[0197] The solubility of the Ubi4 protein was improved by adding a
solubility tail, GKKGK (SEQ ID NO:109), as a C-terminal extension.
The gene for Ubi4-Fn3 was subcloned into the expression vector
pAS45 using PCR. The C-terminal solubilization tag, GKKGK (SEQ ID
NO:109), was incorporated in this step. E. coli BL21 (DE3)
(Novagen) was transformed with the expression vector (pAS45 and its
derivatives). Cells were grown in M9 minimal media and M9 media
supplemented with Bactotryptone (Difco) containing ampicillin (200
.mu.g/ml). For isotopic labeling, .sup.15N NH.sub.4Cl replaced
unlabeled NH.sub.4Cl in the media. 500 ml medium in a 2 liter
baffle flask was inoculated with 10 ml of overnight culture and
agitated at 37.degree. C. IPTG was added at a final concentration
of 1 mM to initiate protein expression when OD (600 nm) reaches
one. The cells were harvested by centrifugation 3 hours after the
addition of IPTG and kept frozen at -70.degree. C. until used.
[0198] Proteins were purified as follows. Cells were suspended in 5
ml/(g cell) of Tris (50 mM, pH 7.6) containing phenylmethylsulfonyl
fluoride (1 mM). Hen egg lysozyme (Sigma) was added to a final
concentration of 0.5 mg/ml. After incubating the solution for 30
minutes at 37.degree. C., it was sonicated three times for 30
seconds on ice. Cell debris was removed by centrifugation.
Concentrated sodium chloride was added to the solution to a final
concentration of 0.5 M. The solution was applied to a Hi-Trap
chelating column (Pharmacia) preloaded with nickel and equilibrated
in the Tris buffer containing sodium chloride (0.5 M). After
washing the column with the buffer, histag-Fn3 was eluted with the
buffer containing 500 mM imidazole. The protein was further
purified using a ResourceS column (Pharmacia) with a NaCl gradient
in a sodium acetate buffer (20 mM, pH 4.6).
[0199] With the GKKGK (SEQ ID NO:109) tail, the solubility of the
Ubi4 protein was increased to over 1 mM at low pH and up to
.about.50 .mu.M at neutral pH. Therefore, further analyses were
performed on Ubi4 with this C-terminal extension (hereafter
referred to as Ubi4-K). It has been reported that the solubility of
a minibody could be significantly improved by addition of three Lys
residues at the N- or C-termini (Bianchi et al., 1994). In the case
of protein Rop, a non-structured C-terminal tail is critical in
maintaining its solubility (Smith et al., 1995).
[0200] Oligomerization states of the Ubi4 protein were determined
using a size exclusion column. The wild-type Fn3 protein was
monomeric at low and neutral pH's. However, the peak of the Ubi4-K
protein was significantly broader than that of wild-type Fn3, and
eluted after the wild-type protein. This suggests interactions
between Ubi4-K and the column material, precluding the use of size
exclusion chromatography to determine the oligomerization state of
Ubi4. NMR studies suggest that the protein is monomeric at low
pH.
[0201] The Ubi4-K protein retained a binding affinity to ubiquitin
as judged by ELISA (FIG. 15d). However, an attempt to determine the
dissociation constant using a biosensor (Affinity Sensors,
Cambridge, U.K.) failed because of high background binding of
Ubi4-K-Fn3 to the sensor matrix. This matrix mainly consists of
dextran, consistent with the observation that interactions between
Ubi4-K interacts with the cross-linked dextran of the size
exclusion column.
Example XVII
Stability Measurements of Monobodies
[0202] Guanidine hydrochloride (GuHCl)-induced unfolding and
refolding reactions were followed by measuring tryptophan
fluorescence. Experiments were performed on a Spectronic AB-2
spectrofluorometer equipped with a motor-driven syringe (Hamilton
Co.). The cuvette temperature was kept at 30.degree. C. The
spectrofluorometer and the syringe were controlled by a single
computer using a home-built interface. This system automatically
records a series of spectra following GuHCl titration. An
experiment started with a 1.5 ml buffer solution containing 5 .mu.M
protein. An emission spectrum (300-400 nm; excitation at 290 nm)
was recorded following a delay (3-5 minutes) after each injection
(50 or 100 .mu.l) of a buffer solution containing GuHCl. These
steps were repeated until the solution volume reached the full
capacity of a cuvette (3.0 ml). Fluorescence intensities were
normalized as ratios to the intensity at an isofluorescent point
which was determined in separate experiments. Unfolding curves were
fitted with a two-state model using a nonlinear least-squares
routine (Santoro & Bolen, 1988). No significant differences
were observed between experiments with delay times (between an
injection and the start of spectrum acquisition) of 2 minutes and
10 minutes, indicating that the unfolding/refolding reactions
reached close to an equilibrium at each concentration point within
the delay times used.
[0203] Conformational stability of Ubi4-K was measured using
above-described GuHCl-induced unfolding method. The measurements
were performed under two sets of conditions; first at pH 3.3 in the
presence of 300 mM sodium chloride, where Ubi4-K is highly soluble,
and second in TBS, which was used for library screening. Under both
conditions, the unfolding reaction was reversible, and we detected
no signs of aggregation or irreversible unfolding. FIG. 16 shows
unfolding transitions of Ubi4-K and wild-type Fn3 with the
N-terminal (his).sub.6 tag and the C-terminal solubility tag. The
stability of wild-type Fn3 was not significantly affected by the
addition of these tags. Parameters characterizing the unfolding
transitions are listed in Table 8.
TABLE-US-00008 TABLE 8 Stability parameters for Ubi4 and wild-type
Fn3 as determined by GuHCl-induced unfolding Protein .DELTA.G.sub.0
(kcal mol.sup.-1) m.sub.G (kcal mol.sup.-1 M.sup.-1) Ubi4 (pH 7.5)
4.8 .+-. 0.1 2.12 .+-. 0.04 Ubi4 (pH 3.3) 6.5 .+-. 0.1 2.07 .+-.
0.02 Wild-type (pH 7.5) 7.2 .+-. 0.2 1.60 .+-. 0.04 Wild-type (pH
3.3) 11.2 .+-. 0.1 2.03 .+-. 0.02 .DELTA.G.sub.0 is the free energy
of unfolding in the absence of denaturant; m.sub.G is the
dependence of the free energy of unfolding on GuHCl concentration.
For solution conditions, see FIG. 4 caption.
Though the introduced mutations in the two loops certainly
decreased the stability of Ubi4-K relative to wild-type Fn3, the
stability of Ubi4 remains comparable to that of a "typical"
globular protein. It should also be noted that the stabilities of
the wild-type and Ubi4-K proteins were higher at pH 3.3 than at pH
7.5.
[0204] The Ubi4 protein had a significantly reduced solubility as
compared to that of wild-type Fn3, but the solubility was improved
by the addition of a solubility tail. Since the two mutated loops
include the only differences between the wild-type and Ubi4
proteins, these loops must be the origin of the reduced solubility.
At this point, it is not clear whether the aggregation of Ubi4-K is
caused by interactions between the loops, or by interactions
between the loops and the invariable regions of the Fn3
scaffold.
[0205] The Ubi4-K protein retained the global fold of Fn3, showing
that this scaffold can accommodate a large number of mutations in
the two loops tested. Though the stability of the Ubi4-K protein is
significantly lower than that of the wild-type Fn3 protein, the
Ubi4 protein still has a conformational stability comparable to
those for small globular proteins. The use of a highly stable
domain as a scaffold is clearly advantageous for introducing
mutations without affecting the global fold of the scaffold. In
addition, the GuHCl-induced unfolding of the Ubi4 protein is almost
completely reversible. This allows the preparation of a correctly
folded protein even when a Fn3 mutant is expressed in a misfolded
form, as in inclusion bodies. The modest stability of Ubi4 in the
conditions used for library screening indicates that Fn3 variants
are folded on the phage surface. This suggests that a Fn3 clone is
selected by its binding affinity in the folded form, not in a
denatured form. Dickinson et al., proposed that Val 29 and Arg 30
in the BC loop stabilize Fn3. Val 29 makes contact with the
hydrophobic core, and Arg 30 forms hydrogen bonds with Gly 52 and
Val 75. In Ubi4-Fn3, Val 29 is replaced with Arg, while Arg 30 is
conserved. The FG loop was also mutated in the library. This loop
is flexible in the wild-type structure, and shows a large variation
in length among human Fn3 domains (Main et al., 1992). These
observations suggest that mutations in the FG loop may have less
impact on stability. In addition, the N-terminal tail of Fn3 is
adjacent to the molecular surface formed by the BC and FG loops
(FIGS. 1 and 17) and does not form a well-defined structure.
Mutations in the N-terminal tail would not be expected to have
strong detrimental effects on stability. Thus, residues in the
N-terminal tail may be good sites for introducing additional
mutations.
Example XVIII
NMR Spectroscopy of Ubi4-Fn3
[0206] Ubi4-Fn3 was dissolved in [.sup.2H]-Gly HCl buffer (20 mM,
pH 3.3) containing NaCl (300 mM) using an Amicon ultrafiltration
unit. The final protein concentration was 1 mM. NMR experiments
were performed on a Varian Unity INOVA 600 spectrometer equipped
with a triple-resonance probe with pulsed field gradient. The probe
temperature was set at 30.degree. C. HSQC, TOCSY-HSQC and
NOESY-HSQC spectra were recorded using published procedures (Kay et
al., 1992; Zhang et al., 1994). NMR spectra were processed and
analyzed using the NMRPipe and NMRView software (Johnson &
Blevins, 1994; Delaglio et al., 1995) on UNIX workstations.
Sequence-specific resonance assignments were made using standard
procedures (Wuthrich, 1986; Clore & Gronenborn, 1991). The
assignments for wild-type Fn3 (Baron et al., 1992) were confirmed
using a .sup.15N-labeled protein dissolved in sodium acetate buffer
(50 mM, pH 4.6) at 30.degree. C.
[0207] The three-dimensional structure of Ubi4-K was characterized
using this heteronuclear NMR spectroscopy method. A high quality
spectrum could be collected on a 1 mM solution of .sup.15N-labeled
Ubi4 (FIG. 17a) at low pH. The linewidth of amide peaks of Ubi4-K
was similar to that of wild-type Fn3, suggesting that Ubi4-K is
monomeric under the conditions used. Complete assignments for
backbone .sup.1H and .sup.15N nuclei were achieved using standard
.sup.1H, .sup.15N double resonance techniques, except for a row of
His residues in the N-terminal (His).sub.6 tag. There were a few
weak peaks in the HSQC spectrum which appeared to originate from a
minor species containing the N-terminal Met residue. Mass
spectroscopy analysis showed that a majority of Ubi4-K does not
contain the N-terminal Met residue. FIG. 17 shows differences in
.sup.1HN and .sup.15N chemical shifts between Ubi4-K and wild-type
Fn3. Only small differences are observed in the chemical shifts,
except for those in and near the mutated BC and FG loops. These
results clearly indicate that Ubi4-K retains the global fold of
Fn3, despite the extensive mutations in the two loops. A few
residues in the N-terminal region, which is close to the two
mutated loops, also exhibit significant chemical differences
between the two proteins. An HSQC spectrum was also recorded on a
50 .mu.M sample of Ubi4-K in TBS. The spectrum was similar to that
collected at low pH, indicating that the global conformation of
Ubi4 is maintained between pH 7.5 and 3.3.
Example XIX
Stabilization of Fn3 Domain by Removing Unfavorable Electrostatic
Interactions on the Protein Surface
Introduction
[0208] Increasing the conformational stability of a protein by
mutation is a major interest in protein design and biotechnology.
The three-dimensional structures of proteins are stabilized by
combination of different types of forces. The hydrophobic effect,
van der Waals interactions and hydrogen bonds are known to
contribute to stabilize the folded state of proteins (Kauzmann, W.
(1959) Adv. Prot. Chem. 14, 1-63; Dill, K. A. (1990) Biochemistry
29, 7133-7155; Pace, C. N., Shirley, B. A., McNutt, M. &
Gajiwala, K. (1996) Faseb J 10, 75-83). These stabilizing forces
primarily originate from residues that are well packed in a
protein, such as those that constitute the hydrophobic core.
Because a change in the protein core would induce a rearrangement
of adjacent moieties, it is difficult to improve protein stability
by increasing these forces without massive computation
(Malakauskas, S. M. & Mayo, S. L. (1998) Nat Struct Biol 5,
470-475). Ion pairs between charged groups are commonly found on
the protein surface (Creighton, T. E. (1993) Proteins: structures
and molecular properties, Freeman, New York), and an ion pair could
be introduced to a protein with small structural perturbations.
However, a number of studies have demonstrated that the
introduction of an attractive electrostatic interaction, such as an
ion pair, on protein surface has small effects on stability
(Dao-pin, S., Sauer, U., Nicholson, H. & Matthews, B. W. (1991)
Biochemistry 30, 7142-7153; Sali, D., Bycroft, M. & Fersht, A.
R. (1991) J. Mol. Biol. 220, 779-788). A large desolvation penalty
and the loss of conformational entropy of amino acid side chains
oppose the favorable electrostatic contribution (Yang, A.-S. &
Honig, B. (1992) Curr. Opin. Struct. Biol. 2, 40-45; Hendsch, Z. S.
& Tidor, B. (1994) Protein Sci. 3, 211-226). Recent studies
demonstrated that repulsive electrostatic interactions on the
protein surface, in contrast, may significantly destabilize a
protein, and that it is possible to improve protein stability by
optimizing surface electrostatic interactions (Loladze, V. V.,
Ibarra-Molero, B., Sanchez-Ruiz, J. M. & Makhatadze, G. I.
(1999) Biochemistry 38, 16419-16423; Perl, D., Mueller, U.,
Heinemann, U. & Schmid, F. X. (2000) Nat Struct Biol 7,
380-383; Spector, S., Wang, M., Carp, S. A., Robblee, J., Hendsch,
Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)
Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,
Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L.,
Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849).
In the present experiments, the inventor improved protein stability
by modifying surface electrostatic interactions.
[0209] During the characterization of monobodies it was found that
these proteins, as well as wild-type FNfn10, are significantly more
stable at low pH than at neutral pH (Koide, A., Bailey, C. W.,
Huang, X. & Koide, S. (1998) J. Mol. Biol. 284, 1141-1151).
These observations indicate that changes in the ionization state of
some moieties in FNfn10 modulate the conformational stability of
the protein, and suggest that it might be possible to enhance the
conformational stability of FNfn10 at neutral pH by adjusting
electrostatic properties of the protein. Improving the
conformational stability of FNfn10 will also have practical
importance in the use of FNfn10 as a scaffold in biotechnology
applications.
[0210] Described below are experiments that detailed
characterization of the pH dependence of FNfn10 stability,
identified unfavorable interactions between side chain carboxyl
groups, and improved the conformational stability of FNfn10 by
point mutations on the surface. The results demonstrate that the
surface electrostatic interactions contribute significantly to
protein stability, and that it is possible to enhance protein
stability by rationally modulating these interactions.
Experimental Procedures
Protein Expression and Purification
[0211] The wild-type protein used for the NMR studies contained
residues 1-94 of FNfn10 (residue numbering is according to FIG.
2(a) of Koide et al. (Koide, A., Bailey, C. W., Huang, X. &
Koide, S. (1998) J. Mol. Biol. 284, 1141-1151)), and additional two
residues (Met-Gln) at the N-terminus (these two residues are
numbered -2 and -1, respectively). The gene coding for the protein
was inserted in pET3a (Novagen, WI). Eschericha coli BL21 (DE3)
transformed with the expression vector was grown in the M9 minimal
media supplemented with .sup.13C-glucose and .sup.15N-ammonium
chloride (Cambridge Isotopes) as the sole carbon and nitrogen
sources, respectively. Protein expression was induced as described
previously (Koide, A., Bailey, C. W., Huang, X. & Koide, S.
(1998) J. Mol. Biol. 284, 1141-1151). After harvesting the cells by
centrifuge, the cells were lysed as described (Koide, A., Bailey,
C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,
1141-1151). After centrifugation, supernatant was dialyzed against
10 mM sodium acetate buffer (pH 5.0), and the protein solution was
applied to a SP-Sepharose FastFlow column (Amersham Pharmacia
Biotech), and FN3 was eluted with a gradient of sodium chloride.
The protein was concentrated using an Amicon concentrator using
YM-3 membrane (Millipore).
[0212] The wild-type protein used for the stability measurements
contained an N-terminal histag (MGSSHHHHHHSSGLVPRGSH) (SEQ ID
NO:114) and residues -2-94 of FNfn10. The gene for FN3 described
above was inserted in pET15b (Novagen). The protein was expressed
and purified as described (Koide, A., Bailey, C. W., Huang, X.
& Koide, S. (1998) J. Mol. Biol. 284, 1141-1151). The wild-type
protein used for measurements of the pH dependence shown in FIG. 22
contained Arg 6 to Thr mutation, which had originally been
introduced to remove a secondary thrombin cleavage site (Koide, A.,
Bailey, C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,
1141-1151). Because Asp 7, which is adjacent to Arg 6, was found to
be critical in the pH dependence of FN3 stability as detailed under
Results, subsequent studies were performed using the wild-type, Arg
6, background. The genes for the D7N and D7K mutants were
constructed using standard polymerase chain reactions, and inserted
in pET15b. These proteins were prepared in the same manner as for
the wild-type protein. .sup.13C, .sup.15N-labeled proteins for
pK.sub.a measurements were prepared as described above, and the
histag moiety was not removed from these proteins.
Chemical Denaturation Measurements
[0213] Proteins were dissolved to a final concentration of 5 .mu.M
in 10 mM sodium citrate buffer at various pH containing 100 mM
sodium chloride. Guanidine HCl (GuHCl)-induce unfolding experiments
were performed as described previously (Koide, A., Bailey, C. W.,
Huang, X. & Koide, S. (1998) J. Mol. Biol. 284, 1141-1151;
Koide, S., Bu, Z., Risal, D., Pham, T.-N., Nakagawa, T., Tamura, A.
& Engelman, D. M. (1999) Biochemistry 38, 4757-4767). GuHCl
concentration was determined using an Abbe refractometer
(Spectronic Instruments) as described (Pace, C. N. & Sholtz, J.
M. (1997) in Protein structure. A practical approach (Creighton, T.
E., Ed.) Vol. pp 299-321, IRL Press, Oxford). Data were analyzed
according to the two-state model as described (Koide, A., Bailey,
C. W., Huang, X. & Koide, S. (1998) J. Mol. Biol. 284,
1141-1151; Santoro, M. M. & Bolen, D. W. (1988) Biochemistry
27, 8063-8068.).
Thermal Denaturation Measurements
[0214] Proteins were dissolved to a final concentration of 5 .mu.M
in 20 mM sodium phosphate buffer (pH 7.0) containing 0.1 or 1 M
sodium chloride or in 20 mM glycine HCl buffer (pH 2.4) containing
0.1 or 1 M sodium chloride. Additionally 6.3 M urea was included in
all solutions to ensure reversibility of the thermal denaturation
reaction. In the absence of urea it was found that denatured FNfn10
adheres to quartz surface, and that the thermal denaturation
reaction was irreversible. Circular dichroism measurements were
performed using a Model 202 spectrometer equipped with a Peltier
temperature controller (Aviv Instruments). A cuvette with a 0.5-cm
pathlength was used. The ellipticity at 227 nm was recorded as the
sample temperature was raised at a rate of approximately 1.degree.
C. per minute. Because of decomposition of urea at high
temperature, the pH of protein solutions tended to shift upward
during an experiment. The pH of protein solution was measured
before and after each thermal denaturation measurement to ensure
that a shift no more than 0.2 pH unit occurred in each measurement.
At pH 2.4, two sections of a thermal denaturation curve
(30-65.degree. C. and 60-95.degree. C.) were acquired from separate
samples, in order to avoid a large pH shift. The thermal
denaturation data were fit with the standard two-state model (Pace,
C. N. & Sholtz, J. M. (1997) in Protein structure. A practical
approach (Creighton, T. E., Ed.) Vol. pp 299-321, IRL Press,
Oxford):
.DELTA.G(T)=.DELTA.H.sub.m(1-T/T.sub.m)-.DELTA.C.sub.P[(T.sub.m-T)+T
ln(T/T.sub.m)]
where .DELTA.G(T) is the Gibbs free energy of unfolding at
temperature T, .DELTA.H.sub.m is the enthalpy change upon unfolding
at the midpoint of the transition, T.sub.m, and .DELTA.C.sub.P is
the heat capacity change upon unfolding. The value for
.DELTA.C.sub.p was fixed at 1.74 kcal mol.sup.-1 K.sup.-1,
according to the approximation of Myers et al. (Myers, J. K., Pace,
C. N. & Scholtz, J. M. (1995) Protein Sci. 4, 2138-2148). Most
of the datasets taken in the presence of 1 M NaCl did not have a
sufficient baseline for the unfolded state, and thus it was assumed
the slope of the unfolded baseline in the presence of 1 M NaCl to
be identical to that determined in the presence of 0.1 M NaCl.
NMR Spectroscopy
[0215] NMR experiments were performed at 30.degree. C. on an INOVA
600 spectrometer (Varian Instruments). The C(CO)NH experiment
(Grzesiek, S., Anglister, J. & Bax, A. (1993) J. Magn. Reson. B
101, 114-119) and the CBCACOHA experiment (Kay, L. E. (1993) J. Am.
Chem. Soc. 115, 2055-2057) were collected on a [.sup.13C,
.sup.15N]-wild-type FNfn10 sample (1 mM) dissolved in 50 mM sodium
acetate buffer (pH 4.6) containing 5% (v/v) deuterium oxide, using
a Varian 5 mm triple resonance probe with pulsed field gradient.
The carboxyl .sup.13C resonances were assigned based on the
backbone .sup.1H, .sup.13C and .sup.15N resonance assignments of
FNfn10 (Baron, M., Main, A. L., Driscoll, P. C., Mardon, H. J.,
Boyd, J. & Campbell, I. D. (1992) Biochemistry 31, 2068-2073).
pH titration of carboxyl resonances were performed on a 0.3 mM
FNfn10 sample dissolved in 10 mM sodium citrate containing 100 mM
sodium chloride and 5% (v/v) deuterium oxide. An 8 mm
triple-resonance, pulse-field gradient probe (Nanolac Corporation)
was used for pH titration. Two-dimensional H(C)CO spectra were
collected using the CBCACOHA pulse sequence as described previously
(McIntosh, L. P., Hand, G., Johnson, P. E., Joshi, M. D., Koerner,
M., Plesniak, L. A., Ziser, L., Wakarchuk, W. W. & Withers, S.
G. (1996) Biochemistry 35, 9958-9966). Sample pH was changed by
adding small aliquots of hydrochloric acid, and pH was measured
before and after taking NMR data. .sup.1H, .sup.15N-HSQC spectra
were taken as described previously (Kay, L. E., Keifer, P. &
Saarinen, T. (1992) J. Am. Chem. Soc. 114, 10663-10665). NMR data
were processed using the NMRPipe package (Delaglio, F., Grzesiek,
S., Vuister, G. W., Zhu, G., Pfeifer, J. & Bax, A. (1995) J.
Biomol. NMR 6, 277-293), and analyzed using the NMRView software
(Johnson, B. A. & Blevins, R. A. (1994) J. Biomol. NMR 4,
603-614).
[0216] NMR titration curves of the carboxyl .sup.13C resonances
were fit to the Henderson-Hasselbalch equation to determine
pK.sub.a's:
.delta.(pH)=(.delta..sub.acid+.delta..sub.base10.sup.(pH-pK.sup.a.sup.))-
/(1+10.sup.(pH-pKa))
where .delta. is the measured chemical shift, .delta..sub.acid is
the chemical shift associated with the protonated state,
.delta..sub.base is the chemical shift associated with the
deprotonated state, and pK.sub.a is the pK.sub.a value for the
residue. Data were also fit to an equation with two ionizable
groups:
.delta.(pH)=(.delta..sub.AH2'+.delta..sub.AH10.sup.(pH-pK.sup.a1.sup.)+.-
delta..sub.A10.sup.(2pH-pK.sup.a1.sup.pK.sup.a2.sup.))/(1+10.sup.(pH-pK.su-
p.a1.sup.)+10.sup.(2pH-pK.sup.a1.sup.-pK.sup.a.sup.))
where .delta..sub.AH2, .delta..sub.AH and .delta..sub.A are the
chemical shifts associated with the fully protonated, singularly
protonated and deprotonated states, respectively, and pK.sub.a1 and
pK.sub.a2 are pK.sub.a's associated with the two ionization steps.
Data fitting was performed using the nonlinear least-square
regression method in the program Igor Pro (WaveMetrix, OR) on a
Macintosh computer.
Results
pH Dependence of FNfn10 Stability
[0217] Previously, it was found that FNfn10 is more stable at
acidic pH than at neutral pH (Koide, A., Bailey, C. W., Huang, X.
& Koide, S. (1998) J. Mol. Biol. 284, 1141-1151). In the
present experiments, the pH dependence of its stability was further
characterized. Because of its high stability, FNfn10 could not be
fully denatured in urea at 30.degree. C. Thus GuHCl-induced
chemical denaturation (FIG. 18) was used. The denaturation reaction
was fully reversible under all conditions tested. In order to
minimize errors caused by extrapolation, the free energy of
unfolding at 4 M GuHCl was used for comparison (FIG. 18). The
stability increased as the pH was lowered, with apparent plateaus
at both ends of the pH range. The pH dependence curve has an
apparent transition midpoint near pH 4. In addition, a gradual
increase in the m value, the dependence of the unfolding free
energy on denaturant concentration was noted. Pace et al. reported
a similar pH dependence of the m value for barnase (Pace, C. N.,
Laurents, D. V. & Erickson, R. E. (1992) Biochemistry 31,
2728-2734). These results indicate that FNfn10 contains
interactions that stabilize the protein at low pH, or those that
destabilize it at neutral pH. The results also suggest that by
identifying and altering the interactions that give rise to the pH
dependence, one may be able to improve the stability of FNfn10 at
neutral pH to a degree similar to that found at low pH.
Determination of pK.sub.a's of the Side Chain Carboxyl Groups in
Wild-Type FNfn10
[0218] The pH dependence of FNfn10 stability suggests that amino
acids with pK.sub.a near 4 are involved in the observed transition.
The carboxyl groups of Asp and Glu generally have pK.sub.a in this
range (Creighton, T. E. (1993) Proteins: structures and molecular
properties, Freeman, New York). It is well known that if a carboxyl
group has unfavorable (i.e. destabilizing) interactions in the
folded state, its pK.sub.a is shifted to a higher value from its
unperturbed value (Yang, A.-S. & Honig, B. (1992) Curr. Opin.
Struct. Biol. 2, 40-45). If a carboxyl group has favorable
interactions in the folded state, it has a lower pK.sub.a. Thus,
the pK.sub.a values of all carboxylates in FNfn10 using
heteronuclear NMR spectroscopy were determined in order to identify
stabilizing and destabilizing interactions involving carboxyl
groups.
[0219] First, the .sup.13C resonance for the carboxyl carbon of
each Asp and Glu residue in FN3 was assigned (FIG. 19). Next, pH
titration of the .sup.13C resonances for these groups was performed
(FIG. 20). Titration curves for Asp 3, 67 and 80, and Glu 38 and 47
could be fit well with the Henderson-Hasselbalch equation with a
single pK.sub.a. The pK.sub.a values for these residues (Table 9)
are either close to or slightly lower than their respective
unperturbed values (3.8-4.1 for Asp, and 4.1-4.6 for Glu (Kuhlman,
B., Luisi, D. L., Young, P. & Raleigh, D. P. (1999)
Biochemistry 38, 4896-4903)), indicating that these carboxyl groups
are involved in neutral or slightly favorable electrostatic
interactions in the folded state.
TABLE-US-00009 TABLE 9 pK.sub.a values for Asp and Glu residues in
FN3.sup.1. Protein Residue Wild-Type D7N D7K E9 3.84, 5.40.sup.2
4.98 4.53 E38 3.79 3.87 3.86 E47 3.94 3.99 3.99 D3 3.66 3.72 3.74
D7 3.54, 5.54.sup.2 -- -- D23 3.54, 5.25.sup.2 3.68 3.82 D67 4.18
4.17 4.14 D80 3.40 3.49 3.48 .sup.1The standard deviations in the
pK.sub.a values are less than 0.05 pH units for those fit with a
single pK.sub.a and less than 0.15 pH unit for those with two
pK.sub.a's. .sup.2Data for E9, D7 and D23 were fit with a
transition curve with two pK.sub.a values.
[0220] The titration curves for Asp 7 and 23, and Glu 9 were fit
better with the Henderson-Hasselbalch equation with two pK.sub.a
values, and one of the two pK.sub.a values for each were shifted
higher than the respective unperturbed values (FIG. 19B). The
titration curves with two apparent pK.sub.a values of these
carboxyl groups may be due to influence of an ionizable group in
the vicinity. In the three-dimensional structure of FNfn10 (Main,
A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I. D.
(1992) Cell 71, 671-678), Asp 7 and 23, and Glu 9 form a patch on
the surface (FIG. 21), with Asp 7 centrally located in the patch.
Thus, it is reasonable to expect that these residues influence each
other's ionization profile. In order to identify which of the three
residues have a highly upshifted pK.sub.a, the H(C)CO spectrum of
the protein in 99% D.sub.2O buffer at pH* 5.0 (direct pH meter
reading) was then collected. Asp 23 and Glu 9 showed larger
deuterium isotope shifts (0.33 and 0.32 ppm, respectively) than Asp
7 (0.18 ppm). These results show that Asp 23 and Glu 9 are
protonated to a greater degree than Asp 7. Thus, we concluded that
Asp 23 and Glu 9 have highly upshifted pK.sub.a's, due to strong
influence of Asp 7.
Mutational Analysis
[0221] The spatial proximity of Asp 7 and 23, and Glu 9 explains
the unfavorable electrostatic interactions in FNfn10 identified in
this study. At low pH where these residues are protonated and
neutral, the repulsive interactions are expected to be mostly
relieved. Thus, it should be possible to improve the stability of
FNfn10 at neutral pH, by removing the electrostatic repulsion
between these three residues. Because Asp 7 is centrally located
among the three residues, it was decided to mutate Asp 7. Two
mutants, D7N and D7K were prepared. The former neutralizes the
negative charge with a residue of virtually identical size. The
latter places a positive charge at residue 7 and increases the size
of the side chain.
[0222] The .sup.1H, .sup.15N-HSQC spectra of the two mutant
proteins were nearly identical to that of the wild-type protein,
indicating that these mutations did not cause large structural
perturbations (data not shown). The degrees of stability of the
mutant proteins were then characterized using thermal and chemical
denaturation measurements. Thermal denaturation measurements were
performed initially with 100 mM sodium chloride, and 6.3 M urea was
included to ensure reversible denaturation and to decrease the
temperature of the thermal transition. All the proteins were
predominantly folded in 6.3 M urea at room temperature. All the
proteins underwent a cooperative transition, and the two mutants
were found to be significantly more stable than the wild type at
neutral pH (FIG. 22 and Table 10). Furthermore, these mutations
almost eliminated the pH dependence of the conformational stability
of FNfn10. These results confirmed that destabilizing interactions
involving Asp 7 in wild-type FNfn10 at neutral pH are the primary
cause of the pH dependence.
TABLE-US-00010 TABLE 10 The midpoint of thermal denaturation (in
.degree. C.) of wild-type and mutant FN3 in the presence of 6.3M
urea. pH 2.4 pH 7.0 Protein 0.1M NaCl 1M NaCl 0.1M NaCl 1M NaCl
wild type 72 82 62 70 D7N 68 82 69 80 D7K 69 77 70 78 The error in
the midpoints for the 0.1M NaCl data is .+-.0.5.degree. C. Because
most of the 1M NaCl data did not have a sufficient baseline for the
denatured state, the error in the midpoints for these data was
estimated to be .+-.2.degree. C.
[0223] The effect of increased sodium chloride concentration on the
conformational stability of the wild type and the two mutant
proteins was next investigated. All proteins were more stable in 1
M sodium chloride than in 0.1 M sodium chloride (FIG. 22). The
increase of the sodium chloride concentration elevated the T.sub.m
of the mutant proteins by approximately 10.degree. C. at both
acidic and neutral pH (Table 10). Remarkably the wild-type protein
was also equally stabilized at both pH, although it contains
unfavorable interactions among the carboxyl groups at neutral pH
but not at acidic pH.
[0224] Chemical denaturation of FNfn10 proteins was monitored using
fluorescence emission from the single Trp residue of FNfn10 (FIG.
23). The free energies of unfolding at pH 6.0 and 4 M GuHCl were
determined to be 1.1 (.+-.0.3), 1.7 (.+-.0.2) and 1.4 (.+-.0.1)
kcal/mol for the wild type, D7N and D7K, respectively, indicating
that the two mutations also increased the conformational stability
against chemical denaturation.
Determination of the pK.sub.a's of the Side Chain Carboxyl Groups
in the Mutant Proteins
[0225] The ionization properties of carboxyl groups in the two
mutant proteins was investigated. The 2D H(C)CO spectra of the
mutant proteins at the high and low ends of the pH titration (pH
.about.7 and .about.1.5, respectively) were nearly identical to the
respective spectra of the wild type, except for the loss of the
cross peaks for Asp 7 (data not shown). This similarity allowed for
an unambiguous assignment of resonances of the mutants, based on
the assignments for wild-type FNfn10. The pH titration experiments
revealed that, except for Glu 9 and Asp 23, the behaviors of Asp
and Glu carboxyl groups are very close to their counterparts in the
wild-type protein (FIG. 24 Panels A, C, D, F and G, and Table 9),
indicating that the two mutations have marginal effects on the
electrostatic environments for these carboxylates. In contrast, the
titration curves for E9 and D23 show significant changes upon
mutation (FIG. 24 Panels B and E). The pK.sub.a of D23 was lowered
by more than 1.6 and 1.4 pH units in the D7N and D7K mutants,
respectively. These results clearly show that the repulsive
interaction between D7 and D23 contributes to the increase in
pK.sub.a of Asp 23 in the wild-type protein, and that it was
eliminated by the neutralization of the negative charge at residue
7. The pK.sub.a of Glu 9 was reduced by 0.4 pH unit by the D7N
mutation, while it was decreased by 0.8 pH units in the D7K mutant.
The greater reduction of Glu 9 pK.sub.a by the D7K mutation
suggests that there is a favorable interaction between Lys 7 and
Glu 9 in this mutant protein.
Discussion
[0226] The present inventor has identified unfavorable
electrostatic interactions in FNfn10, and improved its
conformational stability by mutations on the protein surface. The
results demonstrate that repulsive interactions between like
charges on protein surface significantly destabilize a protein. The
results are also consistent with recent reports by other groups
(Loladze, V. V., Ibarra-Molero, B., Sanchez-Ruiz, J. M. &
Makhatadze, G. I. (1999) Biochemistry 38, 16419-16423; Perl, D.,
Mueller, U., Heinemann, U. & Schmid, F. X. (2000) Nat Struct
Biol 7, 380-383; Spector, S., Wang, M., Carp, S. A., Robblee, J.,
Hendsch, Z. S., Fairman, R., Tidor, B. & Raleigh, D. P. (2000)
Biochemistry 39, 872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R.,
Alston, R. W., Huyghues-Despointes, B. M., Thurlkill, R. L.,
Scholtz, J. M. & Pace, C. N. (1999) Protein Sci 8, 1843-1849),
in which protein stability was improved by eliminating unfavorable
electrostatic interactions on the surface. In these studies,
candidates for mutations were identified by electrostatic
calculations (Loladze, V. V., Ibarra-Molero, B., Sanchez-Ruiz, J M.
& Makhatadze, G. I. (1999) Biochemistry 38, 16419-16423;
Spector, S., Wang, M., Carp, S. A., Robblee, J., Hendsch, Z. S.,
Fairman, R., Tidor, B. & Raleigh, D. P. (2000) Biochemistry 39,
872-879; Grimsley, G. R., Shaw, K. L., Fee, L. R., Alston, R. W.,
Huyghues-Despointes, B. M., Thurlkill, R. L., Scholtz, J. M. &
Pace, C. N. (1999) Protein Sci 8, 1843-1849) or by sequence
comparison of homologous proteins with different stability (Peri,
D., Mueller, U., Heinemann, U. & Schmid, F. X. (2000) Nat
Struct Biol 7, 380-383). The present strategy using pK.sub.a
determination using NMR has both advantages and disadvantages over
the other strategies. The present method directly identifies
residues that destabilize a protein. Also it does not depend on the
availability of the high-resolution structure of the protein of
interest. Electrostatic calculations may have large errors due to
the flexibility of amino acid side chains on the surface, and the
uncertainty in the dielectric constant on the protein surface and
in the protein interior. For example, in the NMR structure of
FNfn10 (Main, A. L., Harvey, T. S., Baron, M., Boyd, J. &
Campbell, I. D. (1992) Cell 71, 671-678), the root mean squared
deviations among 16 model structures for the O.sup..di-elect cons.
atom of Glu residues are 1.2-2.4 .ANG., and those for Lys
N.sup..zeta. atoms are 1.5-3.1 .ANG.. Such uncertainties in atom
position can potentially cause large differences in calculation
results. On the other hand, the present strategy requires the NMR
assignments for carboxyl residues, and NMR measurements over a wide
pH range. Although recent advances in NMR spectroscopy have made it
straightforward to obtain resonance assignments for a small
protein, some proteins may not be sufficiently soluble over the
desired pH range. In addition, knowledge of the pK.sub.a values of
ionizable groups in the denatured state is necessary for accurately
evaluating contributions of individual residues to stability (Yang,
A.-S. & Honig, B. (1992) Curr. Opin. Struct. Biol. 2, 40-45).
Kuhlman et al. (Kuhlman, B., Luisi. D. L., Young, P. & Raleigh,
D. P. (1999) Biochemistry 38, 4896-4903) showed that pK.sub.a's of
carboxylates in the denatured state has a considerably large range
than those obtained from small model compounds. Despite these
limitations, the present method is applicable to many proteins.
[0227] The inventor showed that the unfavorable interactions
involving the carboxyl groups of Asp 7, Glu 9 and Asp23 were no
longer present if these groups are protonated at low pH or if Asp 7
was replaced with Asn or Lys. The similarity in the measured
stability of the mutants and the wild type at low pH (Table 10)
suggests that no other factors significantly contribute to the pH
dependence of FNfn10 stability and that the mutations caused
minimal structural perturbations. The little structural
perturbation was expected, since the carboxyl groups of these three
residues are at least 50% exposed to the solvent, based on the
solvent accessible surface area calculation on the NMR structure
(Main, A. L., Harvey, T. S., Baron, M., Boyd, J. & Campbell, I.
D. (1992) Cell 71, 671-678).
[0228] The difference in thermal stability of the wild-type protein
between acidic and neutral pH persisted in 1 M sodium chloride
(Table 10). Likewise, the wild-type protein exhibited a large
pH-dependence in stability in 4 M GuHCl (FIG. 18). Furthermore,
upon the increase in the sodium chloride concentration from 0.1 to
1.0 M, the T.sub.m of the wild-type and mutant proteins all
increased by .about.10.degree. C., which is in the same magnitude
as the change in T.sub.m of the wild type by the pH shift. These
data indicate that the unfavorable interactions identified in this
study were not effectively shielded in 1 M NaCl or in 4 M GuHCl.
Because the effect of increased sodium chloride was uniform, this
stabilization effect of sodium chloride is likely due to the
nonspecific salting-out effect (Timasheff, S. N. (1992) Curr. Op.
Struct. Biol. 2, 35-39). Other groups also reported little
shielding effect of salts on electrostatic interactions (Perutz, M.
F., Gronenborn, A. M., Clore, G. M., Fogg, J. H. & Shih, D. T.
(1985) J Mol Biol 183, 491-498; Hendsch, Z. S., Jonsson, T., Sauer,
R. T. & Tidor, B. (1996) Biochemistry 35, 7621-7625).
Electrostatic interactions are often thought to diminish with
increasing ionic strength, particularly if the site of interaction
is highly exposed. Accordingly, the present data at neutral pH
(Table 10) showing no difference in the salt sensitivity between
the wild type and the mutants could be interpreted as Asp 7 not
being responsible for destabilizing electrostatic interactions.
Although the reason for this salt insensitivity is not yet clear,
the present results provide a cautionary note on concluding the
presence and absence of electrostatic interactions solely based on
salt concentration dependence.
[0229] The carboxyl triad (Asp 7 and 23, and Glu 9) is highly
conserved in FNfn10 from nine different organisms that were
available in the protein sequence databank at National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov). In these FNfn10
sequences, Asp 9 is conserved except one case where it is replaced
with Asn, and Glu 9 is completely conserved. The position 23 is
either Asp or Glu, preserving the negative charge. As was
discovered in this study, the interactions among these residues are
destabilizing. Thus, their high conservation, despite their
negative effects on stability, suggests that these residues have
functional importance in the biology of fibronectin. In the
structure of a four-FN3 segment of human fibronectin (Leahy, D. J.,
Aukhil, I. & Erickson, H. P. (1996) Cell 84, 155-164), these
residues are not directly involved in interactions with adjacent
domains. Also these residues are located on the opposite face of
FNfn10 from the integrin-binding RGD sequence in the FG loop (FIG.
21). Therefore, it is not clear why these destabilizing residues
are almost completely conserved in FNfn10. In contrast, no other
FN3 domains in human fibronectin contain this carboxyl triad (for a
sequence alignment, see ref Main, A. L., Harvey, T. S., Baron, M.,
Boyd, J. & Campbell, I. D. (1992) Cell 71, 671-678). The
carboxyl triad of FNfn10 may be involved in important interactions
that have not been identified to date.
[0230] Clarke et al. (Clarke, J., Hamill, S. J. & Johnson, C.
M. (1997) J Mol Biol 270, 771-778) reported that the stability of
the third FN3 of human tenascin (TNfn3) increases as pH was
decreased from 7 to 5. Although they could not perform stability
measurements below pH 5 due to protein aggregation, the pH
dependence of TNfn3 resembles that of FNfn10 shown in FIG. 18.
TNfn3 does not contain the carboxylate triad at positions 7, 9 and
23 (Leahy, D. J., Hendrickson, W. A., Aukhil, I. & Erickson, H.
P. (1992) Science 258, 987-991), indicating that the
destabilization of TNfn3 at neutral pH is caused by a different
mechanism from that for FNfn10. A visual inspection of the TNfn3
structure revealed that it has a large number of carboxyl groups,
and that Glu 834 and Asp 850 (numbering according to ref Leahy, D.
J., Hendrickson, W. A., Aukhil, I. & Erickson, H. P. (1992)
Science 258, 987-991) forms a cross-strand pair. It will be
interesting to examine whether altering this pair can increase the
stability of TNfn3.
[0231] In conclusion, a strategy has been described to
experimentally identify unfavorable electrostatic interactions on
the protein surface and improve the protein stability by relieving
such interactions. The present results have demonstrated that
forming a repulsive interaction between carboxyl groups
significantly destabilize a protein. This is in contrast to the
small contributions of forming a solvent-exposed ion pair.
Unfavorable electrostatic interactions on the surface seem quite
common in natural proteins. Therefore, optimization of the surface
electrostatic properties provides a generally applicable strategy
for increasing protein stability (Loladze, V. V., Ibarra-Molero,
B., Sanchez-Ruiz, J. M. & Makhatadze, G. I. (1999) Biochemistry
38, 16419-16423; Perl, D., Mueller, U., Heinemann, U. & Schmid,
F. X. (2000) Nat Struct Biol 7, 380-383; Spector, S., Wang, M.,
Carp, S. A., Robblee, J., Hendsch, Z. S., Fairman, R., Tidor, B.
& Raleigh, D. P. (2000) Biochemistry 39, 872-879; Grimsley, G.
R., Shaw, K. L., Fee, L. R., Alston, R. W., Huyghues-Despointes, B.
M., Thurlkill, R. L., Scholtz, J. M. & Pace, C. N. (1999)
Protein Sci 8, 1843-1849). In addition, repulsive interactions
between carboxylates can be exploited for destabilizing
undesirable, alternate conformations in protein design ("negative
design").
Example XX
An Extension of the Carboxyl-Terminus of the Monobody Scaffold
[0232] The wild-type protein used for stability measurements is
described under Example 19. The carboxyl-terminus of the monobody
scaffold was extended by four amino acid residues, namely, amino
acid residues (Glu-Ile-Asp-Lys) (SEQ ID NO:119), which are the ones
that immediately follow FNfn10 of human fibronectin. The extension
was introduced into the FNfn10 gene using standard PCR methods.
Stability measurements were performed as described under Example
19. The free energy of unfolding of the extended protein was 7.4
kcal mol.sup.-1 at pH 6.0 and 30.degree. C., very close to that of
the wild-type protein (7.7 kcal mol.sup.-1). These results
demonstrate that the C-terminus of the monobody scaffold can be
extended without decreasing its stability.
[0233] The complete disclosure of all patents, patent documents and
publications cited herein are incorporated by reference as if
individually incorporated. The foregoing detailed description and
examples have been given for clarity of understanding only. No
unnecessary limitations are to be understood therefrom. The
invention is not limited to the exact details shown and described
for variations obvious to one skilled in the art will be included
within the invention defined by the claims.
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Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 121 <210> SEQ ID NO 1 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown: Anti-hen egg
lysozyme (HEL) antibody peptide <400> SEQUENCE: 1 Ala Arg Glu
Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly 1 5 10 <210> SEQ
ID NO 2 <211> LENGTH: 17 <212> TYPE: PRT <213>
ORGANISM: Unknown <220> FEATURE: <223> OTHER
INFORMATION: Description of Unknown: Anti-HEL single VH domain
termed VH8 peptide <400> SEQUENCE: 2 Ala Arg Gly Ala Val Val
Ser Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 1 5 10 15 Gly <210>
SEQ ID NO 3 <211> LENGTH: 16 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Tyr Ala
Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile 1 5 10 15
<210> SEQ ID NO 4 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Mutant D1.3-1 peptide <400> SEQUENCE: 4 Tyr Ala Glu
Arg Asp Tyr Arg Leu Asp Tyr Pro Ile 1 5 10 <210> SEQ ID NO 5
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic Mutant
D1.3-2 peptide <400> SEQUENCE: 5 Tyr Ala Val Arg Asp Tyr Arg
Leu Asp Tyr Pro Ile 1 5 10 <210> SEQ ID NO 6 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Mutant D1.3-3 peptide
<400> SEQUENCE: 6 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Ala
Ser Ser Lys Pro Ile 1 5 10 15 <210> SEQ ID NO 7 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Mutant D1.3-4 peptide
<400> SEQUENCE: 7 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Lys
Pro Ile 1 5 10 <210> SEQ ID NO 8 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutant D1.3-5 peptide <400>
SEQUENCE: 8 Tyr Ala Val Arg Asp Tyr Arg Ser Lys Pro Ile 1 5 10
<210> SEQ ID NO 9 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Mutant D1.3-6 peptide <400> SEQUENCE: 9 Tyr Ala Val
Thr Arg Asp Tyr Arg Leu Ser Ser Lys Pro Ile 1 5 10 <210> SEQ
ID NO 10 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant D1.3-7 peptide <400> SEQUENCE: 10 Tyr Ala Val Thr Glu
Arg Asp Tyr Arg Leu Ser Ser Lys Pro Ile 1 5 10 15 <210> SEQ
ID NO 11 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant VH8-1 peptide <400> SEQUENCE: 11 Tyr Ala Val Ala Val
Val Ser Tyr Tyr Ala Met Asp Tyr Pro Ile 1 5 10 15 <210> SEQ
ID NO 12 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant VH8-2 peptide <400> SEQUENCE: 12 Tyr Ala Val Thr Ala
Val Val Ser Tyr Tyr Ala Ser Ser Lys Pro Ile 1 5 10 15 <210>
SEQ ID NO 13 <211> LENGTH: 59 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN1F <400> SEQUENCE: 13 cgggatccca
tatgcaggtt tctgatgttc cgcgtgacct ggaagttgtt gctgcgacc 59
<210> SEQ ID NO 14 <211> LENGTH: 55 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN1R <400> SEQUENCE: 14 taactgcagg
agcatcccag ctgatcagca ggctagtcgg ggtcgcagca acaac 55 <210>
SEQ ID NO 15 <211> LENGTH: 51 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN2F <400> SEQUENCE: 15 ctcctgcagt
taccgtgcgt tattaccgta tcacgtacgg tgaaaccggt g 51 <210> SEQ ID
NO 16 <211> LENGTH: 39 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide FN2R <400> SEQUENCE: 16 gtgaattcct gaaccgggga
gttaccaccg gtttcaccg 39 <210> SEQ ID NO 17 <211>
LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide FN3F
<400> SEQUENCE: 17 aggaattcac tgtacctggt tccaagtcta
ctgctaccat cagcgg 46 <210> SEQ ID NO 18 <211> LENGTH:
38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide FN3R <400>
SEQUENCE: 18 gtatagtcga cacccggttt caggccgctg atggtagc 38
<210> SEQ ID NO 19 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN4F <400> SEQUENCE: 19 cgggtgtcga
ctataccatc actgtatacg ct 32 <210> SEQ ID NO 20 <211>
LENGTH: 55 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide FN4R
<400> SEQUENCE: 20 cgggatccga gctcgctggg ctgtcaccac
ggccagtaac agcgtataca gtgat 55 <210> SEQ ID NO 21 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide FN5F
<400> SEQUENCE: 21 cagcgagctc caagccaatc tcgattaact accgt 35
<210> SEQ ID NO 22 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN5R <400> SEQUENCE: 22 cgggatcctc
gagttactag gtacggtagt taatcga 37 <210> SEQ ID NO 23
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide FN5R' <400> SEQUENCE: 23 cgggatccac
gcgtgccacc ggtacggtag ttaatcga 38 <210> SEQ ID NO 24
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide gene3F <400> SEQUENCE: 24 cgggatccac
gcgtccattc gtttgtgaat atcaaggcca atcg 44 <210> SEQ ID NO 25
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide gene3R <400> SEQUENCE: 25 ccggaagctt
taagactcct tattacgcag tatgttagc 39 <210> SEQ ID NO 26
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide 38TAABg1II <400> SEQUENCE: 26 ctgttactgg
ccgtgagatc taaccagcga gctcca 36 <210> SEQ ID NO 27
<211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide BC3 <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: a, c, t or g <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (23)..(24)
<223> OTHER INFORMATION: a, c, t or g <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 27 gatcagctgg gatgctcctn nknnknnknn knnktattac cgtatcacgt
a 51 <210> SEQ ID NO 28 <211> LENGTH: 57 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide FG2 <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(23)..(24) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(38)..(39) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 28 tgtatacgct gttactggcn nknnknnknn knnknnknnk tccaagccaa
tctcgat 57 <210> SEQ ID NO 29 <211> LENGTH: 47
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide FG3 <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(21)..(22) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(24)..(25) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(27)..(28) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(30)..(31) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 29 ctgtatacgc tgttactggc nnknnknnkn nkccagcgag ctccaag 47
<210> SEQ ID NO 30 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FG4 <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (23)..(24)
<223> OTHER INFORMATION: a, c, t or g <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 30 catcactgta tacgctgtta ctnnknnknn knnknnktcc aagccaatct
c 51 <210> SEQ ID NO 31 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the BC loop of ubiquitin-binding
monobody clone 211 <400> SEQUENCE: 31 Cys Ala Arg Arg Ala 1 5
<210> SEQ ID NO 32 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 211 <400> SEQUENCE: 32 Arg Trp Ile Pro Leu Ala Lys 1 5
<210> SEQ ID NO 33 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 212 <400> SEQUENCE: 33 Cys Trp Arg Arg Ala 1 5
<210> SEQ ID NO 34 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 212 <400> SEQUENCE: 34 Arg Trp Val Gly Leu Ala Trp 1 5
<210> SEQ ID NO 35 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 213 <400> SEQUENCE: 35 Cys Lys His Arg Arg 1 5
<210> SEQ ID NO 36 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 213 <400> SEQUENCE: 36 Phe Ala Asp Leu Trp Trp Arg 1 5
<210> SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 214 <400> SEQUENCE: 37 Cys Arg Arg Gly Arg 1 5
<210> SEQ ID NO 38 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 214 <400> SEQUENCE: 38 Arg Gly Phe Met Trp Leu Ser 1 5
<210> SEQ ID NO 39 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 215 <400> SEQUENCE: 39 Cys Asn Trp Arg Arg 1 5
<210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 215 <400> SEQUENCE: 40 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 41 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 411 <400> SEQUENCE: 41 Ser Arg Leu Arg Arg 1 5
<210> SEQ ID NO 42 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 411 <400> SEQUENCE: 42 Pro Pro Trp Arg Val 1 5
<210> SEQ ID NO 43 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 422 <400> SEQUENCE: 43 Ala Arg Trp Thr Leu 1 5
<210> SEQ ID NO 44 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 422 <400> SEQUENCE: 44 Arg Arg Trp Trp Trp 1 5
<210> SEQ ID NO 45 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 424 <400> SEQUENCE: 45 Gly Gln Arg Thr Phe 1 5
<210> SEQ ID NO 46 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 424 <400> SEQUENCE: 46 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 47 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of WT from library #2 <400>
SEQUENCE: 47 Ala Val Thr Val Arg 1 5 <210> SEQ ID NO 48
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of WT from library #2 <400> SEQUENCE: 48 Arg
Gly Asp Ser Pro Ala Ser 1 5 <210> SEQ ID NO 49 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.1 <400> SEQUENCE: 49 Cys Asn Trp Arg Arg
1 5 <210> SEQ ID NO 50 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.1
<400> SEQUENCE: 50 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 51 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.2 <400>
SEQUENCE: 51 Cys Met Trp Arg Ala 1 5 <210> SEQ ID NO 52
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.2 <400> SEQUENCE: 52 Arg Trp
Gly Met Leu Arg Arg 1 5 <210> SEQ ID NO 53 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.3 <400> SEQUENCE: 53 Ala Arg Met Arg Glu
1 5 <210> SEQ ID NO 54 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.3
<400> SEQUENCE: 54 Arg Trp Leu Arg Gly Arg Tyr 1 5
<210> SEQ ID NO 55 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.4 <400>
SEQUENCE: 55 Cys Ala Arg Arg Arg 1 5 <210> SEQ ID NO 56
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.4 <400> SEQUENCE: 56 Arg Arg
Ala Gly Trp Gly Trp 1 5 <210> SEQ ID NO 57 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.5 <400> SEQUENCE: 57 Cys Asn Trp Arg Arg
1 5 <210> SEQ ID NO 58 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.5
<400> SEQUENCE: 58 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 59 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.6 <400>
SEQUENCE: 59 Arg Trp Arg Glu Arg 1 5 <210> SEQ ID NO 60
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.6 <400> SEQUENCE: 60 Arg His
Pro Trp Thr Glu Arg 1 5 <210> SEQ ID NO 61 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.7 <400> SEQUENCE: 61 Cys Asn Trp Arg Arg
1 5 <210> SEQ ID NO 62 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.7
<400> SEQUENCE: 62 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 63 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.8 <400>
SEQUENCE: 63 Glu Arg Arg Val Pro 1 5 <210> SEQ ID NO 64
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.8 <400> SEQUENCE: 64 Arg Leu
Leu Leu Trp Gln Arg 1 5 <210> SEQ ID NO 65 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.9 <400> SEQUENCE: 65 Gly Arg Gly Ala Gly
1 5 <210> SEQ ID NO 66 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.9
<400> SEQUENCE: 66 Phe Gly Ser Phe Glu Arg Arg 1 5
<210> SEQ ID NO 67 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.11 <400>
SEQUENCE: 67 Cys Arg Trp Thr Arg 1 5 <210> SEQ ID NO 68
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.11 <400> SEQUENCE: 68 Arg Arg
Trp Phe Asp Gly Ala 1 5 <210> SEQ ID NO 69 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.12 <400> SEQUENCE: 69 Cys Asn Trp Arg Arg
1 5 <210> SEQ ID NO 70 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.12
<400> SEQUENCE: 70 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 71 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of WT from library #4 <400>
SEQUENCE: 71 Ala Val Thr Val Arg 1 5 <210> SEQ ID NO 72
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of WT from library #4 <400> SEQUENCE: 72 Gly
Arg Gly Asp Ser 1 5 <210> SEQ ID NO 73 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.1 <400> SEQUENCE: 73 Gly Gln Arg Thr Phe 1 5
<210> SEQ ID NO 74 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.1 <400>
SEQUENCE: 74 Arg Arg Trp Trp Ala 1 5 <210> SEQ ID NO 75
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.2 <400> SEQUENCE: 75 Gly Gln
Arg Thr Phe 1 5 <210> SEQ ID NO 76 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.2 <400> SEQUENCE: 76 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 77 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.3 <400>
SEQUENCE: 77 Gly Gln Arg Thr Phe 1 5 <210> SEQ ID NO 78
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.3 <400> SEQUENCE: 78 Arg Arg
Trp Trp Ala 1 5 <210> SEQ ID NO 79 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.4 <400> SEQUENCE: 79 Leu Arg Tyr Arg Ser 1 5
<210> SEQ ID NO 80 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.4 <400>
SEQUENCE: 80 Gly Trp Arg Trp Arg 1 5 <210> SEQ ID NO 81
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.5 <400> SEQUENCE: 81 Gly Gln
Arg Thr Phe 1 5 <210> SEQ ID NO 82 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.5 <400> SEQUENCE: 82 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 83 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.6 <400>
SEQUENCE: 83 Gly Gln Arg Thr Phe 1 5 <210> SEQ ID NO 84
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.6 <400> SEQUENCE: 84 Arg Arg
Trp Trp Ala 1 5 <210> SEQ ID NO 85 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.7 <400> SEQUENCE: 85 Leu Arg Tyr Arg Ser 1 5
<210> SEQ ID NO 86 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.7 <400>
SEQUENCE: 86 Gly Trp Arg Trp Arg 1 5 <210> SEQ ID NO 87
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.9 <400> SEQUENCE: 87 Leu Arg
Tyr Arg Ser 1 5 <210> SEQ ID NO 88 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.9 <400> SEQUENCE: 88 Gly Trp Arg Trp Arg 1 5
<210> SEQ ID NO 89 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.11 <400>
SEQUENCE: 89 Gly Gln Arg Thr Phe 1 5 <210> SEQ ID NO 90
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.11 <400> SEQUENCE: 90 Arg Arg
Trp Trp Ala 1 5 <210> SEQ ID NO 91 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.12 <400> SEQUENCE: 91 Leu Arg Tyr Arg Ser 1 5
<210> SEQ ID NO 92 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.12 <400>
SEQUENCE: 92 Gly Trp Arg Trp Arg 1 5 <210> SEQ ID NO 93
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of WT from Table 7 <400> SEQUENCE: 93
gcagttaccg tgcgt 15 <210> SEQ ID NO 94 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of WT from
Table 7 <400> SEQUENCE: 94 Ala Val Thr Val Arg 1 5
<210> SEQ ID NO 95 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of WT from Table 7 <400>
SEQUENCE: 95 ggccgtggtg acagcccagc gagc 24 <210> SEQ ID NO 96
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of WT from Table 7 <400> SEQUENCE: 96 Gly Arg
Gly Asp Ser Pro Ala Ser 1 5 <210> SEQ ID NO 97 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone 1 from Table 7 <400> SEQUENCE: 97 tcgaggttgc
ggcgg 15 <210> SEQ ID NO 98 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the BC loop of clone 1 from Table 7
<400> SEQUENCE: 98 Ser Arg Leu Arg Arg 1 5 <210> SEQ ID
NO 99 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the FG loop of clone 1 from Table 7 <400>
SEQUENCE: 99 ccgccgtgga gggtg 15 <210> SEQ ID NO 100
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone 1 from Table 7 <400> SEQUENCE: 100
Pro Pro Trp Arg Val 1 5 <210> SEQ ID NO 101 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone 2 from Table 7 <400> SEQUENCE: 101 ggtcagcgaa
ctttt 15 <210> SEQ ID NO 102 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone 2
from Table 7 <400> SEQUENCE: 102 Gly Gln Arg Thr Phe 1 5
<210> SEQ ID NO 103 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone 2 from Table 7
<400> SEQUENCE: 103 aggcggtggt gggct 15 <210> SEQ ID NO
104 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the FG loop of clone 2 from Table 7 <400>
SEQUENCE: 104 Arg Arg Trp Trp Ala 1 5 <210> SEQ ID NO 105
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone 3 from Table 7 <400> SEQUENCE: 105
gcgaggtgga cgctt 15 <210> SEQ ID NO 106 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone 3
from Table 7 <400> SEQUENCE: 106 Ala Arg Trp Thr Leu 1 5
<210> SEQ ID NO 107 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone 3 from Table 7
<400> SEQUENCE: 107 aggcggtggt ggtgg 15 <210> SEQ ID NO
108 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the FG loop of clone 3 from Table 7 <400>
SEQUENCE: 108 Arg Arg Trp Trp Trp 1 5 <210> SEQ ID NO 109
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
solubility tail peptide <400> SEQUENCE: 109 Gly Lys Lys Gly
Lys 1 5 <210> SEQ ID NO 110 <211> LENGTH: 96
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Fn3 gene polypeptide <400>
SEQUENCE: 110 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
<210> SEQ ID NO 111 <211> LENGTH: 308 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic designed Fn3 gene polynucleotide <400> SEQUENCE:
111 catatgcagg tttctgatgt tccgcgtgac ctggaagttg ttgctgcgac
cccgactagc 60 ctgctgatca gctgggatgc tcctgcagtt accgtgcgtt
attaccgtat cacgtacggt 120 gaaaccggtg gtaactcccc ggttcaggaa
ttcactgtac ctggttccaa gtctactgct 180 accatcagcg gcctgaaacc
gggtgtcgac tataccatca ctgtatacgc tgttactggc 240 cgtggtgaca
gcccagcgag ctccaagcca atctcgatta actaccgtac ctagtaactc 300 gaggatcc
308 <210> SEQ ID NO 112 <211> LENGTH: 96 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic designed Fn3 gene polypeptide <400>
SEQUENCE: 112 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
<210> SEQ ID NO 113 <400> SEQUENCE: 113 000 <210>
SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic fusion peptide <400> SEQUENCE: 114 Met Gly Ser Ser
His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly
Ser His 20 <210> SEQ ID NO 115 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide from clone Plb25.1
<400> SEQUENCE: 115 Gly Gln Arg Thr Phe Arg Arg Trp Trp Ala 1
5 10 <210> SEQ ID NO 116 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide from clone Plb25.4 <400>
SEQUENCE: 116 Leu Arg Tyr Arg Ser Gly Trp Arg Trp Arg 1 5 10
<210> SEQ ID NO 117 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide from clone pLB24.1 <400> SEQUENCE: 117 Cys
Asn Trp Arg Arg Arg Ala Tyr Arg Tyr Trp Arg 1 5 10 <210> SEQ
ID NO 118 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide from clone pLB24.3 <400> SEQUENCE: 118 Ala Arg Met
Arg Glu Arg Trp Leu Arg Gly Arg Tyr 1 5 10 <210> SEQ ID NO
119 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 119 Glu Ile Asp Lys 1
<210> SEQ ID NO 120 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Anti-hen egg lysozyme
(HEL) antibody peptide <400> SEQUENCE: 120 Arg Asp Tyr Arg 1
<210> SEQ ID NO 121 <211> LENGTH: 96 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 121
Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5
10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val
Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 121
<210> SEQ ID NO 1 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Anti-hen egg lysozyme
(HEL) antibody peptide <400> SEQUENCE: 1 Ala Arg Glu Arg Asp
Tyr Arg Leu Asp Tyr Trp Gly Gln Gly 1 5 10 <210> SEQ ID NO 2
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Unknown <220> FEATURE: <223> OTHER INFORMATION:
Description of Unknown: Anti-HEL single VH domain termed VH8
peptide <400> SEQUENCE: 2 Ala Arg Gly Ala Val Val Ser Tyr Tyr
Ala Met Asp Tyr Trp Gly Gln 1 5 10 15 Gly <210> SEQ ID NO 3
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 Tyr Ala Val Thr Gly Arg Gly
Asp Ser Pro Ala Ser Ser Lys Pro Ile 1 5 10 15 <210> SEQ ID NO
4 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant D1.3-1 peptide <400> SEQUENCE: 4 Tyr Ala Glu Arg Asp
Tyr Arg Leu Asp Tyr Pro Ile 1 5 10 <210> SEQ ID NO 5
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic Mutant
D1.3-2 peptide <400> SEQUENCE: 5 Tyr Ala Val Arg Asp Tyr Arg
Leu Asp Tyr Pro Ile 1 5 10 <210> SEQ ID NO 6 <211>
LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Mutant D1.3-3 peptide
<400> SEQUENCE: 6 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Ala
Ser Ser Lys Pro Ile 1 5 10 15 <210> SEQ ID NO 7 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Mutant D1.3-4 peptide
<400> SEQUENCE: 7 Tyr Ala Val Arg Asp Tyr Arg Leu Asp Tyr Lys
Pro Ile 1 5 10 <210> SEQ ID NO 8 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutant D1.3-5 peptide <400>
SEQUENCE: 8 Tyr Ala Val Arg Asp Tyr Arg Ser Lys Pro Ile 1 5 10
<210> SEQ ID NO 9 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Mutant D1.3-6 peptide <400> SEQUENCE: 9 Tyr Ala Val
Thr Arg Asp Tyr Arg Leu Ser Ser Lys Pro Ile 1 5 10 <210> SEQ
ID NO 10 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant D1.3-7 peptide <400> SEQUENCE: 10 Tyr Ala Val Thr Glu
Arg Asp Tyr Arg Leu Ser Ser Lys Pro Ile 1 5 10 15 <210> SEQ
ID NO 11 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant VH8-1 peptide <400> SEQUENCE: 11 Tyr Ala Val Ala Val
Val Ser Tyr Tyr Ala Met Asp Tyr Pro Ile 1 5 10 15 <210> SEQ
ID NO 12 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutant VH8-2 peptide <400> SEQUENCE: 12 Tyr Ala Val Thr Ala
Val Val Ser Tyr Tyr Ala Ser Ser Lys Pro Ile 1 5 10 15 <210>
SEQ ID NO 13 <211> LENGTH: 59 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN1F <400> SEQUENCE: 13 cgggatccca
tatgcaggtt tctgatgttc cgcgtgacct ggaagttgtt gctgcgacc 59
<210> SEQ ID NO 14 <211> LENGTH: 55 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN1R <400> SEQUENCE: 14 taactgcagg
agcatcccag ctgatcagca ggctagtcgg ggtcgcagca acaac 55 <210>
SEQ ID NO 15 <211> LENGTH: 51 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN2F <400> SEQUENCE: 15 ctcctgcagt
taccgtgcgt tattaccgta tcacgtacgg tgaaaccggt g 51 <210> SEQ ID
NO 16 <211> LENGTH: 39 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide FN2R <400> SEQUENCE: 16 gtgaattcct gaaccgggga
gttaccaccg gtttcaccg 39 <210> SEQ ID NO 17 <211>
LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide FN3F
<400> SEQUENCE: 17 aggaattcac tgtacctggt tccaagtcta
ctgctaccat cagcgg 46
<210> SEQ ID NO 18 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN3R <400> SEQUENCE: 18 gtatagtcga
cacccggttt caggccgctg atggtagc 38 <210> SEQ ID NO 19
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide FN4F <400> SEQUENCE: 19 cgggtgtcga ctataccatc
actgtatacg ct 32 <210> SEQ ID NO 20 <211> LENGTH: 55
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide FN4R <400>
SEQUENCE: 20 cgggatccga gctcgctggg ctgtcaccac ggccagtaac agcgtataca
gtgat 55 <210> SEQ ID NO 21 <211> LENGTH: 35
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide FN5F <400>
SEQUENCE: 21 cagcgagctc caagccaatc tcgattaact accgt 35 <210>
SEQ ID NO 22 <211> LENGTH: 37 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FN5R <400> SEQUENCE: 22 cgggatcctc
gagttactag gtacggtagt taatcga 37 <210> SEQ ID NO 23
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide FN5R' <400> SEQUENCE: 23 cgggatccac
gcgtgccacc ggtacggtag ttaatcga 38 <210> SEQ ID NO 24
<211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide gene3F <400> SEQUENCE: 24 cgggatccac
gcgtccattc gtttgtgaat atcaaggcca atcg 44 <210> SEQ ID NO 25
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide gene3R <400> SEQUENCE: 25 ccggaagctt
taagactcct tattacgcag tatgttagc 39 <210> SEQ ID NO 26
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide 38TAABg1II <400> SEQUENCE: 26 ctgttactgg
ccgtgagatc taaccagcga gctcca 36 <210> SEQ ID NO 27
<211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide BC3 <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (20)..(21) <223> OTHER
INFORMATION: a, c, t or g <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (23)..(24)
<223> OTHER INFORMATION: a, c, t or g <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 27 gatcagctgg gatgctcctn nknnknnknn knnktattac cgtatcacgt
a 51 <210> SEQ ID NO 28 <211> LENGTH: 57 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide FG2 <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(23)..(24) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(38)..(39) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 28 tgtatacgct gttactggcn nknnknnknn knnknnknnk tccaagccaa
tctcgat 57 <210> SEQ ID NO 29 <211> LENGTH: 47
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide FG3 <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(21)..(22) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(24)..(25) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(27)..(28) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(30)..(31) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 29 ctgtatacgc tgttactggc nnknnknnkn nkccagcgag ctccaag 47
<210> SEQ ID NO 30 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide FG4 <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (23)..(24)
<223> OTHER INFORMATION: a, c, t or g <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(29)..(30) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, t or g <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, t or g <400>
SEQUENCE: 30 catcactgta tacgctgtta ctnnknnknn knnknnktcc aagccaatct
c 51 <210> SEQ ID NO 31 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the BC loop of ubiquitin-binding
monobody clone 211 <400> SEQUENCE: 31 Cys Ala Arg Arg Ala 1 5
<210> SEQ ID NO 32 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 211 <400> SEQUENCE: 32 Arg Trp Ile Pro Leu Ala Lys 1 5
<210> SEQ ID NO 33 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 212 <400> SEQUENCE: 33 Cys Trp Arg Arg Ala 1 5
<210> SEQ ID NO 34 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 212 <400> SEQUENCE: 34 Arg Trp Val Gly Leu Ala Trp 1 5
<210> SEQ ID NO 35 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 213 <400> SEQUENCE: 35 Cys Lys His Arg Arg 1 5
<210> SEQ ID NO 36 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 213 <400> SEQUENCE: 36 Phe Ala Asp Leu Trp Trp Arg 1 5
<210> SEQ ID NO 37 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 214 <400> SEQUENCE: 37 Cys Arg Arg Gly Arg 1 5
<210> SEQ ID NO 38 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 214 <400> SEQUENCE: 38 Arg Gly Phe Met Trp Leu Ser 1 5
<210> SEQ ID NO 39 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 215 <400> SEQUENCE: 39 Cys Asn Trp Arg Arg 1 5
<210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 215 <400> SEQUENCE: 40 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 41 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 411 <400> SEQUENCE: 41 Ser Arg Leu Arg Arg 1 5
<210> SEQ ID NO 42 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 411 <400> SEQUENCE: 42 Pro Pro Trp Arg Val 1 5
<210> SEQ ID NO 43 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 422 <400> SEQUENCE: 43 Ala Arg Trp Thr Leu 1 5
<210> SEQ ID NO 44 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of ubiquitin-binding monobody
clone 422 <400> SEQUENCE: 44 Arg Arg Trp Trp Trp 1 5
<210> SEQ ID NO 45 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of ubiquitin-binding monobody
clone 424
<400> SEQUENCE: 45 Gly Gln Arg Thr Phe 1 5 <210> SEQ ID
NO 46 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the FG loop of ubiquitin-binding monobody clone 424
<400> SEQUENCE: 46 Arg Arg Trp Trp Ala 1 5 <210> SEQ ID
NO 47 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of WT from library #2 <400> SEQUENCE:
47 Ala Val Thr Val Arg 1 5 <210> SEQ ID NO 48 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the FG
loop of WT from library #2 <400> SEQUENCE: 48 Arg Gly Asp Ser
Pro Ala Ser 1 5 <210> SEQ ID NO 49 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB24.1 <400> SEQUENCE: 49 Cys Asn Trp Arg Arg 1 5
<210> SEQ ID NO 50 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB24.1 <400>
SEQUENCE: 50 Arg Ala Tyr Arg Tyr Arg Trp 1 5 <210> SEQ ID NO
51 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone pLB24.2 <400> SEQUENCE: 51
Cys Met Trp Arg Ala 1 5 <210> SEQ ID NO 52 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the FG
loop of clone pLB24.2 <400> SEQUENCE: 52 Arg Trp Gly Met Leu
Arg Arg 1 5 <210> SEQ ID NO 53 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB24.3 <400> SEQUENCE: 53 Ala Arg Met Arg Glu 1 5
<210> SEQ ID NO 54 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB24.3 <400>
SEQUENCE: 54 Arg Trp Leu Arg Gly Arg Tyr 1 5 <210> SEQ ID NO
55 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone pLB24.4 <400> SEQUENCE: 55
Cys Ala Arg Arg Arg 1 5 <210> SEQ ID NO 56 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the FG
loop of clone pLB24.4 <400> SEQUENCE: 56 Arg Arg Ala Gly Trp
Gly Trp 1 5 <210> SEQ ID NO 57 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB24.5 <400> SEQUENCE: 57 Cys Asn Trp Arg Arg 1 5
<210> SEQ ID NO 58 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB24.5 <400>
SEQUENCE: 58 Arg Ala Tyr Arg Tyr Arg Trp 1 5 <210> SEQ ID NO
59 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone pLB24.6 <400> SEQUENCE: 59
Arg Trp Arg Glu Arg 1 5 <210> SEQ ID NO 60 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the FG
loop of clone pLB24.6 <400> SEQUENCE: 60 Arg His Pro Trp Thr
Glu Arg 1 5 <210> SEQ ID NO 61 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB24.7 <400> SEQUENCE: 61 Cys Asn Trp Arg Arg 1 5
<210> SEQ ID NO 62 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB24.7 <400> SEQUENCE: 62 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 63 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.8 <400>
SEQUENCE: 63 Glu Arg Arg Val Pro 1 5 <210> SEQ ID NO 64
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.8 <400> SEQUENCE: 64 Arg Leu
Leu Leu Trp Gln Arg 1 5 <210> SEQ ID NO 65 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.9 <400> SEQUENCE: 65 Gly Arg Gly Ala Gly
1 5 <210> SEQ ID NO 66 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.9
<400> SEQUENCE: 66 Phe Gly Ser Phe Glu Arg Arg 1 5
<210> SEQ ID NO 67 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB24.11 <400>
SEQUENCE: 67 Cys Arg Trp Thr Arg 1 5 <210> SEQ ID NO 68
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB24.11 <400> SEQUENCE: 68 Arg Arg
Trp Phe Asp Gly Ala 1 5 <210> SEQ ID NO 69 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the BC
loop of clone pLB24.12 <400> SEQUENCE: 69 Cys Asn Trp Arg Arg
1 5 <210> SEQ ID NO 70 <211> LENGTH: 7 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic sequence of the FG loop of clone pLB24.12
<400> SEQUENCE: 70 Arg Ala Tyr Arg Tyr Arg Trp 1 5
<210> SEQ ID NO 71 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of WT from library #4 <400>
SEQUENCE: 71 Ala Val Thr Val Arg 1 5 <210> SEQ ID NO 72
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of WT from library #4 <400> SEQUENCE: 72 Gly
Arg Gly Asp Ser 1 5 <210> SEQ ID NO 73 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.1 <400> SEQUENCE: 73 Gly Gln Arg Thr Phe 1 5
<210> SEQ ID NO 74 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.1 <400>
SEQUENCE: 74 Arg Arg Trp Trp Ala 1 5 <210> SEQ ID NO 75
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.2 <400> SEQUENCE: 75 Gly Gln
Arg Thr Phe 1 5 <210> SEQ ID NO 76 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.2 <400> SEQUENCE: 76 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 77 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.3 <400>
SEQUENCE: 77 Gly Gln Arg Thr Phe 1 5 <210> SEQ ID NO 78
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.3 <400> SEQUENCE: 78 Arg Arg
Trp Trp Ala 1 5
<210> SEQ ID NO 79 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.4 <400>
SEQUENCE: 79 Leu Arg Tyr Arg Ser 1 5 <210> SEQ ID NO 80
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.4 <400> SEQUENCE: 80 Gly Trp
Arg Trp Arg 1 5 <210> SEQ ID NO 81 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.5 <400> SEQUENCE: 81 Gly Gln Arg Thr Phe 1 5
<210> SEQ ID NO 82 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.5 <400>
SEQUENCE: 82 Arg Arg Trp Trp Ala 1 5 <210> SEQ ID NO 83
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.6 <400> SEQUENCE: 83 Gly Gln
Arg Thr Phe 1 5 <210> SEQ ID NO 84 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.6 <400> SEQUENCE: 84 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 85 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.7 <400>
SEQUENCE: 85 Leu Arg Tyr Arg Ser 1 5 <210> SEQ ID NO 86
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.7 <400> SEQUENCE: 86 Gly Trp
Arg Trp Arg 1 5 <210> SEQ ID NO 87 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of clone
pLB25.9 <400> SEQUENCE: 87 Leu Arg Tyr Arg Ser 1 5
<210> SEQ ID NO 88 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone pLB25.9 <400>
SEQUENCE: 88 Gly Trp Arg Trp Arg 1 5 <210> SEQ ID NO 89
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone pLB25.11 <400> SEQUENCE: 89 Gly Gln
Arg Thr Phe 1 5 <210> SEQ ID NO 90 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone
pLB25.11 <400> SEQUENCE: 90 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 91 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone pLB25.12 <400>
SEQUENCE: 91 Leu Arg Tyr Arg Ser 1 5 <210> SEQ ID NO 92
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone pLB25.12 <400> SEQUENCE: 92 Gly Trp
Arg Trp Arg 1 5 <210> SEQ ID NO 93 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the BC loop of WT from
Table 7 <400> SEQUENCE: 93 gcagttaccg tgcgt 15 <210>
SEQ ID NO 94 <211> LENGTH: 5 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of WT from Table 7 <400>
SEQUENCE: 94 Ala Val Thr Val Arg 1 5 <210> SEQ ID NO 95
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of WT from Table 7 <400> SEQUENCE: 95
ggccgtggtg acagcccagc gagc 24
<210> SEQ ID NO 96 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of WT from Table 7 <400>
SEQUENCE: 96 Gly Arg Gly Asp Ser Pro Ala Ser 1 5 <210> SEQ ID
NO 97 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone 1 from Table 7 <400>
SEQUENCE: 97 tcgaggttgc ggcgg 15 <210> SEQ ID NO 98
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone 1 from Table 7 <400> SEQUENCE: 98 Ser
Arg Leu Arg Arg 1 5 <210> SEQ ID NO 99 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone 1
from Table 7 <400> SEQUENCE: 99 ccgccgtgga gggtg 15
<210> SEQ ID NO 100 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the FG loop of clone 1 from Table 7
<400> SEQUENCE: 100 Pro Pro Trp Arg Val 1 5 <210> SEQ
ID NO 101 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone 2 from Table 7 <400>
SEQUENCE: 101 ggtcagcgaa ctttt 15 <210> SEQ ID NO 102
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the BC loop of clone 2 from Table 7 <400> SEQUENCE: 102
Gly Gln Arg Thr Phe 1 5 <210> SEQ ID NO 103 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic sequence of the FG
loop of clone 2 from Table 7 <400> SEQUENCE: 103 aggcggtggt
gggct 15 <210> SEQ ID NO 104 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone 2
from Table 7 <400> SEQUENCE: 104 Arg Arg Trp Trp Ala 1 5
<210> SEQ ID NO 105 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic sequence of the BC loop of clone 3 from Table 7
<400> SEQUENCE: 105 gcgaggtgga cgctt 15 <210> SEQ ID NO
106 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
sequence of the BC loop of clone 3 from Table 7 <400>
SEQUENCE: 106 Ala Arg Trp Thr Leu 1 5 <210> SEQ ID NO 107
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic sequence
of the FG loop of clone 3 from Table 7 <400> SEQUENCE: 107
aggcggtggt ggtgg 15 <210> SEQ ID NO 108 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic sequence of the FG loop of clone 3
from Table 7 <400> SEQUENCE: 108 Arg Arg Trp Trp Trp 1 5
<210> SEQ ID NO 109 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic solubility tail peptide <400> SEQUENCE: 109 Gly Lys
Lys Gly Lys 1 5 <210> SEQ ID NO 110 <211> LENGTH: 96
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Fn3 gene polypeptide <400>
SEQUENCE: 110 Met Gln Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Asp Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
<210> SEQ ID NO 111 <211> LENGTH: 308 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic designed Fn3 gene polynucleotide <400> SEQUENCE:
111 catatgcagg tttctgatgt tccgcgtgac ctggaagttg ttgctgcgac
cccgactagc 60 ctgctgatca gctgggatgc tcctgcagtt accgtgcgtt
attaccgtat cacgtacggt 120
gaaaccggtg gtaactcccc ggttcaggaa ttcactgtac ctggttccaa gtctactgct
180 accatcagcg gcctgaaacc gggtgtcgac tataccatca ctgtatacgc
tgttactggc 240 cgtggtgaca gcccagcgag ctccaagcca atctcgatta
actaccgtac ctagtaactc 300 gaggatcc 308 <210> SEQ ID NO 112
<211> LENGTH: 96 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic designed
Fn3 gene polypeptide <400> SEQUENCE: 112 Met Gln Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Gly Arg 65 70 75 80 Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile
Asn Tyr Arg Thr 85 90 95 <210> SEQ ID NO 113 <400>
SEQUENCE: 113 000 <210> SEQ ID NO 114 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic fusion peptide <400> SEQUENCE:
114 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15 Arg Gly Ser His 20 <210> SEQ ID NO 115 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide from clone
Plb25.1 <400> SEQUENCE: 115 Gly Gln Arg Thr Phe Arg Arg Trp
Trp Ala 1 5 10 <210> SEQ ID NO 116 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide from clone Plb25.4
<400> SEQUENCE: 116 Leu Arg Tyr Arg Ser Gly Trp Arg Trp Arg 1
5 10 <210> SEQ ID NO 117 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide from clone pLB24.1 <400>
SEQUENCE: 117 Cys Asn Trp Arg Arg Arg Ala Tyr Arg Tyr Trp Arg 1 5
10 <210> SEQ ID NO 118 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide from clone pLB24.3 <400>
SEQUENCE: 118 Ala Arg Met Arg Glu Arg Trp Leu Arg Gly Arg Tyr 1 5
10 <210> SEQ ID NO 119 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
119 Glu Ile Asp Lys 1 <210> SEQ ID NO 120 <211> LENGTH:
4 <212> TYPE: PRT <213> ORGANISM: Unknown <220>
FEATURE: <223> OTHER INFORMATION: Description of Unknown:
Anti-hen egg lysozyme (HEL) antibody peptide <400> SEQUENCE:
120 Arg Asp Tyr Arg 1 <210> SEQ ID NO 121 <211> LENGTH:
96 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 121 Met Gln Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp
Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val
Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80
Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85
90 95
* * * * *