U.S. patent application number 13/622978 was filed with the patent office on 2013-04-04 for phenolic binding peptides.
This patent application is currently assigned to DANISCO US INC.. The applicant listed for this patent is Danisco US Inc.. Invention is credited to Giselle G. Janssen, Christopher J. Murray, Pilar Tijerina, Franciscus J.C. Van Gastel.
Application Number | 20130084618 13/622978 |
Document ID | / |
Family ID | 32093987 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130084618 |
Kind Code |
A1 |
Murray; Christopher J. ; et
al. |
April 4, 2013 |
PHENOLIC BINDING PEPTIDES
Abstract
The present application relates to peptides which bind to
tannin, polyphenolic or anthocyanin compounds, and particularly to
tea and wine stains on a fabric or other surface. The invention
also concerns binding peptide conjugates which includes a binding
peptide coupled to an agent and the use of the binding peptide
conjugate for delivering an agent to a desired target.
Inventors: |
Murray; Christopher J.;
(Soquel, CA) ; Tijerina; Pilar; (Austin, TX)
; Van Gastel; Franciscus J.C.; (Union City, CA) ;
Janssen; Giselle G.; (Dixon, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Danisco US Inc.; |
Palo Alto |
CA |
US |
|
|
Assignee: |
DANISCO US INC.
Palo Alto
CA
|
Family ID: |
32093987 |
Appl. No.: |
13/622978 |
Filed: |
September 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12144572 |
Jun 23, 2008 |
8293702 |
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13622978 |
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10528514 |
Dec 9, 2005 |
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PCT/US03/31776 |
Oct 6, 2003 |
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12144572 |
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60417210 |
Oct 8, 2002 |
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Current U.S.
Class: |
435/188 ;
435/264; 530/327; 530/329 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
7/06 20130101; A61K 38/00 20130101; Y02A 50/30 20180101; C07K
2319/20 20130101; Y02A 50/473 20180101 |
Class at
Publication: |
435/188 ;
530/329; 435/264; 530/327 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Claims
1. A binding peptide comprising an amino acid sequence shown in any
one of SEQ ID NOs. 1-316 and a binding peptide having at least 70%
sequence identity thereto.
2. (canceled)
3. The binding peptide of claim 1, wherein the peptide is selected
from the group consisting of KTPSPHG (SEQ ID NO. 1); PNTTRHS (SEQ
ID NO. 2); LWTSPQL (SEQ ID NO. 8); TNNTSPT (SEQ ID NO. 24); SPTSTNS
(SEQ ID NO. 43); TTTTPFA (SEQ ID NO. 77); SWNTSPL (SEQ ID NO. 80);
QAVKASHATMYL (SEQ ID NO. 97); SYDLIPPRSGLA (SEQ ID NO. 104);
DPNTTSH (SEQ ID NO.118); KASHLVP (SEQ ID NO: 132); LPTSTLT (SEQ ID
NO. 139); QNQKSTT (SEQ ID NO. 158); SIIPPRQ (SEQ ID NO. 168);
WSNKPLSPNDLR (SEQ ID NO. 193) and peptides having at least 75%
amino acid sequence identity thereto.
4. The binding peptide of claim 1, having a repeatable motif
selected from the group consisting of LPL (SEQ ID NOs. 120, 123,
115 and 250); FAT (SEQ ID NOs. 125, 227 and 235); STT (SEQ ID NOs.
90, 158, 230 and 310); HSP (SEQ ID NOs. 18, 252 and 307); TNK (SEQ
ID NOs. 40, 259 and 287); SPL (SEQ ID NOs. 53, 80, 152, 229, 232
and 292); THS (SEQ ID NOs. 62, 209 and 290); TSP (SEQ ID NOs. 8,
24, 80, 223 and 291); SPT (SEQ ID NOs. 24, 43 and 266); AQT (SEQ ID
NOs. 59, 134 and 205); NSS (SEQ ID NOs. 31, 86, 213, 227 and 278);
PAL (SEQ ID NOs. 109, 224 and 256); SGL (SEQ ID NOs. 104, 284 and
298); and TQT (SEQ ID NOs. 105, 281 and 287) and a binding peptide
having the repeatable motif and at least 75% amino acid sequence
identity to a binding peptide having the repeatable motif and
listed herein.
5. The binding peptide of claim 1, wherein said peptide binds to a
compound selected from the group consisting of tannin, anthocyanin
and phenolic compounds.
6. The binding peptide of claim 1, wherein said peptide binds to a
tea or wine stain.
7. The binding peptide of claim 6, wherein the peptide binds to a
tea or wine stain on a fabric.
8. The binding peptide of claim 6, wherein the peptide binds to a
tea or wine stain on a surface.
9. The binding peptide of claim 8, wherein the surface is selected
from the group consisting of ceramic, glass, wood, paper, skin,
hair and plastic.
10. The binding peptide of claim 1, further comprising a cysteine
amino acid residue at the N and C terminus of said peptide.
11. A binding peptide conjugate comprising a binding peptide of
claim 1, linked to an agent.
12. (canceled)
13. The conjugate according to claim 11, wherein said agent is a
protein.
14. The conjugate of claim 13, wherein the protein is an
enzyme.
15. The conjugate of claim 14, wherein said enzyme is an enzyme
that catalyzes an oxidation-reduction reaction and is selected from
the group consisting of laccases, phenol oxidases, catalases,
bilirubrin oxidases, glucose oxidases, and peroxidases.
16. The conjugate of claim 11, wherein said binding peptide is
covalently linked to said agent.
17. The conjugate of claim 11, wherein said binding peptide and
said agent are separated by a linker.
18-25. (canceled)
26. A method for delivering an agent to a target comprising a)
conjugating an agent with a binding peptide of claim 1 to form a
binding peptide conjugate and b) exposing a target to the binding
peptide conjugated, wherein the binding peptide conjugate binds to
said target.
27. The method according to claim 26, wherein the target is a tea
or wine stain.
28. The method according to claim 26, wherein the agent is an
enzyme.
29-35. (canceled)
Description
[0001] The present patent application is a continuation application
of U.S. patent application Ser. No. 10/528,514, filed 9 Dec. 2005,
which was a national stage entry of international patent
application PCT/US03/31776, filed 6 Oct. 2003, which claimed
priority of U.S. patent application Ser. No. 60/417,210, filed 8
Oct. 2002.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to novel binding peptides and
to binding peptide conjugates, wherein the binding peptide is
linked to an agent. In particular the peptides bind to tannin,
polyphenolic and/or anthocyanin compounds and more particularly to
tea and wine stains. The invention also concerns the use of the
binding peptides for delivering agents to targeted tannin,
polyphenolic and/or anthocyanin compounds that comprise tea and
wine stains.
[0003] Binding peptides and proteins conjugated to a binding
peptide have numerous uses in many varied applications. Some of
these uses include applications in enzymatic compositions,
particularly detergent compositions, in personal care applications,
in food industry applications, in diagnostic applications and
therapeutic applications.
[0004] For example oxidative-reductase (redox) enzymes capable of
modifying the color associated with colored compounds could be used
more effectively if conjugated to a peptide that targeted a
particular compound. For example, a peptide that binds to a tannin,
polyphenolic or anthocyanin compound as a target on a fabric or on
a surface such as ceramic could deliver the redox enzyme more
effectively to the specific target and result in more effective
bleaching of the stain. This selective targeting of a tannin,
polyphenolic or anthocyanin compound can provide a significant
improvement in the cleaning performance of enzymatic compositions.
In another example, a peptide that binds to a tannin, polyphenolic
or anthocyanin compound on a surface such as skin, teeth or nails
could deliver the redox enzyme more effectively to the specifically
targeted pigmented areas which then may result in bleaching of the
area.
SUMMARY OF THE INVENTION
[0005] In a first aspect the invention concerns a peptide which
binds to a compound selected from the group consisting of tannin,
anthocyanin and phenolic compounds. In one preferred embodiment the
binding peptide of the invention will bind to a tea or wine stain
and particularly to a tea or wine stain on a fabric or on a surface
such as ceramic, glass, wood, paper, metal, plastic, skin, teeth,
hair or nails.
[0006] In a second aspect the invention relates to a binding
peptide comprising an amino acid sequence shown in any one of SEQ
ID NOs. 1-316 and a binding peptide having at least 70% sequence
identity thereto. In a further aspect the invention relates to a
binding peptide consisting essentially of an amino acid sequence
shown in any one of SEQ ID NOs. 1-316 and a binding peptide having
at least 70% sequence identity thereto. In one embodiment the
binding peptides of the invention further comprise a cysteine amino
acid residue at the N and C terminus of a peptide as shown in any
one of SEQ ID NOs. 1-316 or a binding peptide having at least 70%
sequence identity thereto. In a another embodiment, the binding
peptide is selected from the group consisting of KTPSPHG (SEQ ID
NO. 1); PNTTRHS (SEQ ID NO. 2); LWTSPQL (SEQ ID NO. 8); TNNTSPT
(SEQ ID NO. 24); SPTSTNS (SEQ ID NO. 43); TTTTPFA (SEQ ID NO. 77);
SWNTSPL (SEQ ID NO. 80); QAVKASHATMYL (SEQ ID NO. 97); SYDLIPPRSGLA
(SEQ ID NO. 104); DPNTTSH (SEQ ID NO.118); KASHLVP (SEQ ID NO:
132); LPTSTLT (SEQ ID NO. 139); QNQKSTT (SEQ ID NO. 158); SIIPPRQ
(SEQ ID NO. 168); SNKPLSPNDLR (SEQ ID NO. 193) and peptides having
at least 75% amino acid sequence identity thereto.
[0007] In a third aspect the invention concerns a binding peptide
having a repeatable motif selected from the group consisting of LPL
(SEQ ID NOs. 120, 123, 115 and 250); FAT (SEQ ID NOs. 125, 227 and
235); STT (SEQ ID NOs. 90, 158, 230 and 310); HSP (SEQ ID NOs. 18,
252 and 307); TNK (SEQ ID NOs. 40, 259 and 287); SPL (SEQ ID NOs.
53, 80, 152, 229, 232 and 292); THS (SEQ ID NOs. 62, 209 and 290);
TSP (SEQ ID NOs. 8, 24, 80, 223 and 291); SPT (SEQ ID NOs. 24, 43
and 266); AQT (SEQ ID NOs. 59, 134 and 205); NSS (SEQ ID NOs. 31,
86, 213, 227 and 278); PAL (SEQ ID NOs. 109, 224 and 256); SGL (SEQ
ID NOs. 104, 284 and 298); and TQT (SEQ ID NOs. 105, 281 and 287)
and a binding peptide having at least 75% amino acid sequence
identity thereto.
[0008] In a fourth aspect the invention concerns a binding peptide
conjugate which comprises a binding peptide of the invention linked
to an agent. In one embodiment the agent is a protein. In a
preferred embodiment the protein is an enzyme and particularly an
enzyme that catalyzes an oxidation-reduction reaction. In a
particularly preferred embodiment the enzyme is selected from the
group consisting of laccases, phenol oxidases, catalases, bilirubin
oxidases, glucose oxidases and peroxidases. In one embodiment the
binding peptide is covalently linked to said agent and in another
embodiment the binding peptide and said agent are separated by a
linker.
[0009] In a fifth aspect the invention relates to an enzymatic
composition which comprises a binding peptide of the invention, an
enzyme, and one or more surfactants. In one embodiment the
composition is a detergent composition. In a second embodiment the
enzymatic composition comprises a) a binding peptide conjugate
which includes a binding peptide of the invention linked to an
agent, wherein the agent is an enzyme and b) one or more
surfactants. In a third embodiment the invention relates to a
method for modifying a tea or wine stain on a fabric or a surface
comprising contacting the fabric or surface having a tea or wine
stain thereon with the enzymatic composition. Preferably the
surface is ceramic, skin or teeth. Modification may include either
removing the tea or wine stain or enhancing the tea or wine
stain.
[0010] In a sixth aspect the invention relates to a method for
delivering an agent to a target which comprises conjugating an
agent with a binding peptide of the invention to form a binding
peptide conjugate and exposing a target to the binding peptide
conjugated, wherein the binding peptide conjugate binds to said
target. In one embodiment the target is a tea or wine stain. In
another embodiment the agent is an enzyme. In another embodiment
the target is a tea or wine stain.
[0011] In a seventh aspect the invention relates to polynucleotide
sequences encoding a binding peptide or a binding peptide conjugate
according to the invention and to vectors and host cells comprising
said polynucleotide sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1B illustrate the amino acid sequences of peptides
represented by SEQ ID NOs. 1-111 that bind to tea stains on cotton.
A peptide string having an amino acid residue designated as X in a
specific position indicates that the amino acid residue is not
known and may be any L-amino acid.
[0013] FIGS. 2A-2B illustrate the amino acid sequences of peptides
represented by SEQ ID NOs. 112-201 that bind to tea stains on
ceramic. A peptide string having an amino acid residue designated
as X in a specific position indicates that the amino acid residue
is not known and may be any L-amino acid.
[0014] FIGS. 3A, 3B and 3C illustrate the amino acid sequences of
peptides represented by SEQ ID NOs. (202-316) that bind to wine
stains on cotton. A peptide string having an amino acid residue
designated as X in a specific position indicates that the amino
acid residue is not known and may be any L-amino acid.
[0015] FIG. 4 illustrates the preferential binding of phage bound
peptides (PNTTRHS (SEQ ID NO. 2); LWTSPQL (SEQ ID NO. 8); TNNTSPT
(SEQ ID NO. 24); SYGPMTN (SEQ ID NO. 65); LHQNQKS (SEQ ID NO. 68);
and SWNTSPL (SEQ ID NO. 80)) to tea stains on cotton swatches
(.box-solid.) compared to binding on non-stained cotton swatches
(.quadrature.). WT is a control, a phage without a binding peptide
insert.
DETAILED DESCRIPTION OF THE INVENTION
General Terms
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. For the
purpose of the present invention, the following terms are used to
describe the invention herein.
[0017] The term "peptide" refers to an oligomer in which the
monomer units are amino acids (typically, but not limited to
L-amino acids) linked by an amide bond. Peptides may be two or more
amino acids in length. Peptides that are greater than 100 amino
acids in length are generally referred to as polypeptides. However,
the terms, peptide, polypeptide and protein may be used
interchangeably. Standard abbreviations for amino acids are used
herein and reference is made to Singleton et al., (1987) DICTIONARY
OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2ND ED. page 35.
[0018] The term "isolated" as used herein refers to a nucleic acid
or amino acid sequence that is removed from at least one component
with which it is naturally associated.
[0019] "Percent sequence identity" with respect to peptide or
polynucleotide sequences refers to the percentage of residues that
are identical in the two sequences. Thus 95% amino acid sequence
identity means that 95% of the amino acids in the sequences are
identical. Percent identity can be determined by direct comparison
of the sequence information provided between two sequences and can
be determined by various commercially available computer programs
such as BESTFIT, FASTA, DNASTAR, TFASTA and BLAST.
[0020] A "binding peptide" according to the invention is a peptide
that binds to a target with a binding affinity of at least about
10.sup.-2M, at least about 10.sup.-3M, at least about 10.sup.-4 M,
at least about 10.sup.-5 M and preferably between about 10.sup.-2 M
to 10.sup.-15 M, between about 10.sup.-2M to 10.sup.-10 M and
between about 10.sup.-2M to 10.sup.-9 M.
[0021] The binding affinity of a peptide for its target or the
binding affinity of a binding peptide conjugate for its target may
be described by the dissociation constant (K.sub.D). K.sub.D is
defined by k.sub.off/k.sub.on. The k.sub.off value defines the rate
at which a bound-target complex breaks apart or separates. This
term is sometimes referred to in the art as the kinetic stability
of the peptide-target complex or the ratio of any other measurable
quantity that reflects the ratio of binding affinity such as an
enzyme-linked immunosorbent assay (ELISA) signal. K.sub.on
describes the rate at which the target and the binding peptide (or
binding peptide conjugate) combine to form a bound-target complex.
In one aspect, the k.sub.off value for the bound-target complex
will be less that about 10.sup.-2 sec.sup.-1, less that about
10.sup.-3 sec.sup.-1, less than about 10.sup.-4 sec.sup.-1 and also
less than about 10.sup.-5 sec.sup.-1.
[0022] The term "conjugation" as used herein means an agent is
chemically linked or joined directly or indirectly to a terminus of
a binding peptide. The phrases "binding peptide conjugate" and
"conjugated agent" are used interchangeably herein. A binding
peptide conjugate or a conjugated agent may be considered a fusion
protein. A fusion protein refers to a protein that comprises two
separate and distinct regions that may or may not originate from
the same protein.
[0023] An "agent" is any molecule or compound that is capable of
being conjugated with a binding peptide of the invention and
preferably capable of be chaperoned or delivered to a target.
Agents according to the invention comprise a broad class of
compounds including but not limited to proteins, carbohydrates,
lipids, chemicals, such as dyes, bleaching compounds and
fluorescent compounds, and ions, such as salts.
[0024] Selectivity is defined herein as enhanced binding of a
binding peptide to a target compared to the binding of the peptide
to a non-target. Selectivity may also be defined as the enhanced
binding of a conjugated agent to a target compared to the binding
of a non-conjugated agent to the same target. Selectivity may be in
the range of about 1.25:1 to 25:1; about 1.5:1 to 15:1; about 1.5:1
to 10:1; and about 1.5:1 to 5:1. Preferably the selectively is at
least 4:1, 3:1 or 2:1 for either a) the binding of a binding
peptide to a target compared to the binding of the peptide to a
non-target or b) the binding of a conjugated agent to a target
compared to the binding of the non-conjugated agent to the same
target.
[0025] Preferred targets of a binding peptide or a binding peptide
conjugate of the invention are tannin, phenolic or anthocyanin
compounds. Particularly tannin, phenolic or anthocyanin compounds
found in tea or wine, and particularly a tea and/or wine stain.
However, the target compounds may be found on a material, surface
or solution.
[0026] A stain is defined herein as a colored compound which
undergoes a redox chemical reaction when exposed to certain classes
of enzymes, for example phenol oxidizing enzymes such as laccases.
A coloured compound is a substance that adds colour to a textile or
to substances which result in the visual appearances of stains.
Targeted classes of coloured substances which may appear as a stain
include the following;
[0027] a) porphyrin derived structures, such as heme in blood stain
or chlorophyll in plants;
[0028] b) tannins and polyphenols (see P. Ribereau-Gayon, Plant
Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972, pp.169-198)
which occur in tea, wine, coffee, chocolate, cola, banana and peach
stains;
[0029] c) carotenoids and carotenoid derivatives, which are the
red, orange and yellow pigments occurring in fruits and vegetables
such as tomato, mango, carrots, paprika and leafy green vegetables.
Commonly known carotenoids include alpha and beta-carotene,
lycopene, lutein, zeaxanthin, and cryptoxantin. These compounds
include the oxygenated carotenoids, xanthophylls. Reference is made
to G. E. Bartley et al., The Plant Cell (1995), Vol. 7, 1027-1038,
Biochemical Nomenclature and Related Documents, 2nd Ed. Portland
Press (1992), pages 226-238, and Pure Appl. Chem, (1974)
41:407-431);
[0030] d) anthocyanins, the highly coloured molecules which occur
in many fruits and flowers, such as red grapes, cranberries,
blueberries and cherries and red wine (P. Ribereau-Gayon, Plant
Phenolics, Ed. Oliver & Boyd, Edinburgh, 1972, 135-169);
and
[0031] e) Maillard reaction products, the yellow/brown coloured
substances which appear upon heating of mixtures of carbohydrate
molecules in the presence of protein/peptide structures, such as
found in cooking oil.
[0032] A coloured compound may also be a dye that may be
incorporated into a fiber by chemical reaction, adsorption or
dispersion. Examples include direct Blue dyes, acid Blue dyes,
reactive Blue dyes, and reactive Black dyes.
[0033] A stain may occur on a fabric or other surface material.
Nonlimiting examples of fabric include, cotton, wool, silk,
polyester, rayon, linen, nylon and blends thereof. Nonlimiting
examples of a surface material include, ceramic, glass, wood,
paper, metal, plastic, stainless steel, teeth, bone, nails, skin
and hair.
[0034] The phrase "modify the colour associated with a coloured
compound" means that the coloured compound is changed through
oxidation-reduction, either directly or indirectly, such that the
colour appears modified i.e. the colour visually appears to be
increased; decreased; changed from one color to another, such as
from blue to red; decoloured; bleached; or removed; particularly
bleached.
[0035] As used in the specification and claims, the singular "a",
"an" and "the" include the plural references unless the context
clearly dictates otherwise. For example, the term a host cell may
include a plurality of host cells.
[0036] The following references describe the general techniques
employed herein: Sambrook et al (1989) MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbour Laboratory Press, Cold
Spring Harbour, N.Y.; and Ausubel et al. (1987) CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, Greene-Publishing & Wiley Interscience NY
(Supplemented through 1999). The contents of all references,
patents and published patent applications cited throughout this
application are hereby incorporated by reference in their
entirety.
B. Binding Peptides
[0037] The binding peptides of the invention may be obtained and
identified using methods well known in the art. These methods may
include the use of random peptide libraries, synthetic peptide
libraries, peptide loop libraries, antibody libraries and protein
libraries. Many of these library collections are commercially
available. Screening techniques may include yeast display, ribosome
display, biopanning and acid elution. Once a library is screened,
the peptides that bind to a specific target may be identified by
various well-known means in the art including but not limited to
acid elution, polymerase chain reaction (PCR), sequencing, and the
like. These techniques are described in various references such as
Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA 87:6378; Parmley
et al., (1988) Gene 73:305; Balass et al., (1996) Anal. Biochem.,
243:264; Huls et al., (1996) Nature Biotechnol. 7:276 and WO
01/79479).
[0038] A typical method for selecting binding peptides of the
invention involves removing from a library those peptides that bind
non-specifically to a material and then incubating the remaining
members of the library with a stained material containing the
target substrate.
[0039] Once selected a binding peptide may be identified, amplified
or produced in bulk by any one of a number of standard techniques.
For example the peptide may be produced recombinantly using genetic
engineering or the peptide may be chemically synthesized.
[0040] Preferably the binding peptides of the invention are between
4 and 50 amino acids in length, also between 4-25 amino acids in
length, between 4-20 amino acids in length and between 6-15 amino
acids in length.
[0041] The binding peptides according to the invention include the
peptides listed in FIGS. 1A-1B (SEQ ID NOs. 1-111), FIGS. 2A-2B
(SEQ ID NOs. 112-201); and FIGS. 3A, 3B and 3C (SEQ ID NOs.
202-316. These peptides bind to molecules found in tea and/or
wine.
[0042] The invention further includes binding peptides having at
least 60% but less than 100% amino acid sequence identity to a
binding peptide listed in FIG. 1A, 1B, 2A, 2B, 3A, 3B or 3C (SEQ ID
NOs. 1-316). For example at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 93%, at least
95%, at least 97%, at least 99% amino acid sequence identity. A
peptide having at least 60% sequence identity to a binding peptide
listed in FIG. 1A, 1B, 2A, 2B, 3A, 3B or 3C will also have a
binding affinity for the same target in the range of 10.sup.-2M to
10.sup.-15M, generally at least about 10.sup.-2M, at least about
10.sup.-3M, at least about 10.sup.-4M and at least about
10.sup.-5M. In one embodiment a binding peptide according to the
invention will have no more than 2 amino acid residues that differ
from a binding 7-mer peptide which is listed in FIG. 1A, 1B, 2A,
2B, 3A, 3B or 3C. In another embodiment, a binding peptide
according to the invention will have no more than 3 amino acid
residues that differ from a binding 12-mer peptide which is listed
in FIG. 1A, 1B, 2A, 2B, 3A, 3B or 3C.
[0043] In one embodiment, preferred binding peptides of FIGS. 1A-1B
are: KTPSPHG (SEQ ID NO. 1); PNTTRHS (SEQ ID NO. 2); KTPSSME (SEQ
ID NO. 5); LWTSPQL (SEQ ID NO. 8); SLNNTNT (SEQ ID NO. 11); QKHSPGH
(SEQ ID NO. 18); TNNTSPT (SEQ ID NO. 24); QTQPPGS (SEQ ID NO. 25);
TMAPAKN (SEQ ID NO. 36); SHLDKRL (SEQ ID NO. 37); TTTNKPL (SEQ ID
NO. 40); SPTSTNS (SEQ ID NO. 43); PGSNATQ (SEQ ID NO. 44); SQDTPMY
(SEQ ID NO. 45); TDPSMMN (SEQ ID NO. 46); GQADRLQ (SEQ ID NO. 47);
TPQRLLT (SEQ ID NO. 48); SQMSPLH (SEQ ID NO. 53); TQNPTHS (SEQ ID
NO. 62); HGSSAHP (SEQ ID NO. 64); TTAAPQM (SEQ ID NO. 70); SSNLPFA
(SEQ ID NO. 71); TTTTPFA (SEQ ID NO. 77); SWNTSPL (SEQ ID NO. 80);
PSPPTNQ (SEQ ID NO. 82); PLTSTQP (SEQ ID NO. 85); HVSDLAG (SEQ ID
NO. 87); TLSRTTA (SEQ ID NO. 88); HLRSTTD (SEQ ID NO. 90); SPMQPRL
(SEQ ID NO. 93); FTANLRA (SEQ ID NO. 94); LFLPPTPPPEPL (SEQ ID NO.
96); QAVKASHATMYL (SEQ ID NO. 97); ETQPSAMGGSSL (SEQ ID NO. 99);
STSWPPQPHLSP (SEQ ID NO. 102); SYDLIPPRSGLA (SEQ ID NO. 104);
NTTQTLRHVSLA (SEQ ID NO. 105); TSGFDRALSPSL (SEQ ID NO. 107);
SNSTMNALAPA (SEQ ID NO. 111) and peptides having at least 70% amino
acid sequence identity thereto.
[0044] Particularly preferred binding peptides of FIGS. 1A and 1B
are PNTTRHS (SEQ ID NO. 2); LWTSPQL (SEQ ID NO. 8); TNNTSPT (SEQ ID
NO. 24); and SWNTSPL (SEQ ID NO. 80) and peptides having at least
75% amino acid sequence identity thereto.
[0045] In another embodiment, preferred binding peptides of FIGS.
2A-2B are:
[0046] ALGXIPXTAHQW (SEQ ID NO. 114); ARSIQPF (SEQ ID NO. 115);
ATVILTD (SEQ ID NO. 116); DPNTTSH (SEQ ID NO. 118); FLPLLTL (SEQ ID
NO. 120); FQLIPTG (SEQ ID NO. 121); GVPFATP (SEQ ID NO. 125);
IPTTRQT (SEQ ID NO. 131); KASHLVP (SEQ ID NO. 132); KDPSWPSQAQTP
(SEQ ID NO. 134); LPTSTLT (SEQ ID NO. 139); PPSPLTP (SEQ ID NO.
152); PTLAGAS (SEQ ID NO. 154); QDTAPLT (SEQ ID NO. 157); QNQKSTT
(SEQ ID NO. 158); QPGHLDI (SEQ ID NO. 159); LSLPMQ (SEQ ID NO.
164); SIIPPRQ (SEQ ID NO. 168); SSLLPRS (SEQ ID NO. 174); TAPLISI
(SEQ ID NO. 177); TKTTWQT (SEQ ID NO. 180); TLFYTKX (SEQ ID NO.
181); TQRLTTH (SEQ ID NO. 182); TSLVPDK (SEQ ID NO. 184); WQLARPK
(SEQ ID NO. 191); WQTXLTD (SEQ ID NO. 192); WSNKPLSPNDLR (SEQ ID
NO. 193); YTKTSQY (SEQ ID NO. 201); and peptides having at least
70% amino acid sequence identity thereto.
[0047] In another embodiment preferred binding peptides of FIGS.
1A, 1B, 2A and 2B include KTPSPHG (SEQ ID NO. 1); PNTTRHS (SEQ ID
NO. 2); LWTSPQL (SEQ ID NO. 8); TNNTSPT (SEQ ID NO. 24); SPTSTNS
(SEQ ID NO. 43); TTTTPFA (SEQ ID NO. 77); SWNTSPL (SEQ ID NO. 80);
QAVKASHATMYL (SEQ ID NO. 97); SYDLIPPRSGLA (SEQ ID NO. 104);
DPNTTSH (SEQ ID NO.118); KASHLVP (SEQ ID NO: 132); LPTSTLT (SEQ ID
NO. 139); QNQKSTT (SEQ ID NO. 158); SIIPPRQ (SEQ ID NO. 168);
WSNKPLSPNDLR (SEQ ID NO. 193) and peptides having at least 75%
amino acid sequence identity thereto.
[0048] In another embodiment, preferred binding peptides of FIGS.
3A, 3B and 3C are: QYHGPLP (SEQ ID NO. 203); TGNSSQQ (SEQ ID NO.
213); LPLQPLMPPLNQ (SEQ ID NO. 225); NSSPFATMPNAL (SEQ ID NO. 227);
NVNNHIH (SEQ ID NO. 247); ADRLRPT (SEQ ID NO. 251); HSPQMQS (SEQ ID
NO. 252); SPALVNS (SEQ ID NO. 256); TNKIPPL (SEQ ID NO. 259);
TNPNHIM (SEQ ID NO. 260); QPLKTKQ (SEQ ID NO. 262); TKSPTAI (SEQ ID
NO. 266); KSPEYPF (SEQ ID NO. 270); TTQTNKD (SEQ ID NO. 287);
PATNPNH (SEQ ID NO. 289); SPLYHDR (SEQ ID NO. 292); NAFESLF (SEQ ID
NO. 296); DPQANLT (SEQ ID NO. 299); RQANLTQ (SEQ ID NO. 300);
LDQHSMK, (SEQ ID NO. 301); PSTTKHG (SEQ ID NO. 310); and peptides
having at least 70% amino acid sequence identity thereto.
[0049] In a further embodiment, the binding peptides according to
the invention may include cysteine residues on each end of the
peptide. These binding peptides are more specifically referred to
herein as binding peptide C-C derivatives. The cysteine residues
form a disulfide bridge, making the peptide form a loop on the
surface of the phage. Thus, if the binding peptide is used as an
internal replacement or insert for protein loops or turns, the
binding peptide may be used in the C-C derivative form or the non
C-C derivative form. Particularly preferred C-C derivative peptides
are those comprising 7 amino acids. In one aspect preferred C-C
derivatives are the preferred 7-mers disclosed in FIGS. 1A-1B;
FIGS. 2A-2B and FIGS. 3A, 3B and 3C as designated above.
[0050] Additionally, a linker (L) molecule (also sometimes referred
to as a spacer moiety in the prior art) may be added to either end
of a binding peptide (P), for example, L-P or P-L. The linker
molecule may enhance the binding of the peptide to its target. A
linker molecule may be any carbon containing compound, such as a
short peptide, for example, the amino acid triad GGH or GGHGG; a
carbon chain, for example, (CH.sub.2), wherein n equals 1 to 10; a
polymer, for example PEG (CH.sub.2--O), wherein n equals 2-20; a
sugar; a lipid or the like.
[0051] As stated above, the linker molecule may be attached to the
binding peptide alone or the linker molecule may be part of the
binding peptide conjugate. For example, when the linker (L) is
placed between the binding peptide (P) and the agent (A), (A-L-P)
or when the linker is attached to the peptide at the non-conjugated
end, (A-P-L). A linker molecule may be attached to any of the
binding peptides represented as SEQ ID NOs. 1-316 of FIGS. 1A, 1B,
2A, 2B, 3A, 3B and 3C.
[0052] Repeatable motifs have been observed in a number of the
binding peptides listed in FIGS. 1A, 1B, 2A, 2B, 3A, 3B and 3C.
Repeatable motifs include at least three consecutive amino acid
residues in a peptide string and may include four, five or six
consecutive amino acid residues that are found in at least two of
the binding peptides listed in FIGS. 1A, 1B, 2A, 2B, 3A, 3B and
3C.
[0053] Preferred repeatable motifs which are included in binding
peptides listed in FIGS. 1A and 1B, which bind to a tea stain are:
INAQ, KTPS, NSSS, NTSP, SNAT, GSS, HQT, PGS, SSS, TPQ, TQP, TSP,
TTA, TTT, APA, HQG, HPS, HVS, KPL, LNN, LPF, SNS, SPL, SPM, SPT,
SRL, LSP, LSR, MMN, MYL, NAQ, NNT, NPT, NTT, PAK, PFA, PLH, PPP,
PPT, PQM, PSL, PSP, PTH, QKH, RLQ, SLA, SNA, TQK, TQM, TQN, TSG,
TST, STM, STR, TAA, TDP, TMA, TTP, TTQ, VTT, AND QNQ.
[0054] Particularly preferred repeatable motifs and peptides which
include these motifs of FIGS. 1A and 1B are INAQ, (SEQ ID NOs. 59
and 84); KTPS, (SEQ ID NOs. 1 and 5); NSSS, (SEQ ID NOs. 31 and
86); NTSP, (SEQ ID NOs. 24 and 80); SNAT, (SEQ ID NOs. 32 and 44);
GSS, (SEQ ID NOs. 64, 99 and 100); HQT, (SEQ ID NOs. 7, 15 and
108); PGS, (SEQ ID NOs. 25, 44 and 100); TPQ, (SEQ ID NOs. 14, 48
and 66); TQP, (SEQ ID NOs. 25, 85 and 99); TSP, (SEQ ID NOs. 8, 24
and 80) and TTA, (SEQ ID NOs. 28, 70 and 88).
[0055] Preferred repeatable motifs which are included in binding
peptides listed in FIGS. 2A and 2B which bind to a tea stain are:
ATP, APL, HPP, IPT, ISI, KTSQ, LPR, LPM, LPL, LPT, LST, LTD, LTP,
LVP, LSP, PLI, PPR, PAP, PTL, PLT, SLV, SWP, TSQ, TAPL, TLF, TLT,
TKT, WQT, and YTK.
[0056] Particularly preferred repeatable motifs and peptides which
include these motifs of FIGS. 2A and 2B are LTP, (SEQ ID NOs. 142,
152 and 163) and LSP, (SEQ ID NOs. 137, 176 and 193).
[0057] Preferred repeatable motifs which are included in binding
peptides listed in FIGS. 3A, 3B and 3C which bind to a wine stain
are: QANLT; TNPNH; ANLT; NPNH; QANL; TNPN; PPL; SPL; AAT; ANL; DRL;
ELP; FAT; GLS; HAM; HGP; HQA; HSP; KSP; KTK; LHD; LPL; LPP; LYH;
MPN; MQS; NAF; NHI; NLT; NMN; NPN; NTL; NVN; NSS; PAL; PAT; PHP;
PLM; PLN; PLP; PNH; PTA; PYT; QPL; QTN; RLH; RSA; SGL; SHS; SLF;
SPQ; SRS; STP; STS; STT; TAE; TFA; TGN; THS; TKH; TNK; TNP; TPP;
TPR; TQT; TRS; TSP; TTI; VNS; WNA; and YPF.
[0058] Particularly preferred repeatable motifs and peptides which
include these motifs of FIGS. 3A, 3B and 3C are QANLT (SEQ ID NOs.
299 and 300); TNPNH (SEQ ID NOs. 260 and 289); ANLT (SEQ ID NOs.
300 and 299); NPNH (SEQ ID NOs. 260 and 289); QANL (SEQ ID NOs. 299
and 300); TNPN (SEQ ID NOs. 260 and 289); NSS (SEQ ID NOs, 213, 227
and 278); PPL (SEQ ID NOs. 225, 229 and 259) and SPL (SEQ ID NOs.
229, 232 and 292).
[0059] Preferred repeatable motifs for peptides that bind to
compounds in wine and tea and binding peptides including these
repeatable motifs are the following: LPL (SEQ ID NOs. 120, 123, 115
and 250); FAT (SEQ ID NOs. 125, 227 and 235); STT (SEQ ID NOs. 90,
158, 230 and 310); HSP (SEQ ID NOs. 18, 252 and 307); TNK (SEQ ID
NOs. 40, 259 and 287); SPL (SEQ ID NOs. 53, 80, 152, 229, 232 and
292); THS (SEQ ID NOs. 62, 209 and 290); TSP (SEQ ID NOs. 8, 24,
80, 223 and 291); SPT (SEQ ID NOs. 24, 43 and 266); AQT (SEQ ID
NOs. 59, 134 and 205); NSS (SEQ ID NOs. 31, 86, 213, 227 and 278);
PAL (SEQ ID NOs. 109, 224 and 256); SGL (SEQ ID NOs. 104, 284 and
298); and TQT (SEQ ID NOs. 105, 281 and 287).
[0060] The repeatable motif may also include a cysteine residue at
the beginning and/or end of the motif, non-limiting examples
include (C)SPM, (C)SPL, (C)KTPS, (C)TTT, TTA(C) and the like.
[0061] In general, the repeatable motifs may occur alone in a
binding peptide, as multiple motifs in the same binding peptide, in
sequential order, or overlapping one another. For example the
binding peptide KTPSPHG (SEQ ID NO: 1) includes the repeatable
motif KTPS. The binding peptide LGTPQQT (SEQ ID NO: 14) includes
the repeatable motif TPQ. The binding peptides RQANLTQ (SEQ ID NO.
300) and DPQANLT (SEQ ID NO. 299) include the repeatable motif
QANLT. The binding peptides TTAAPQM (SEQ ID NO. 70) and
ETQPSAMGGSSL (SEQ ID NO. 99) include two repeatable motifs, in the
same sequence. The binding peptide LPLQPLMPPLNQ (SEQ ID NO. 225)
includes two repeatable motifs LPL and QPL in sequential order.
[0062] Peptides, other than the binding peptides illustrated in
FIGS. 1A,1B, 2A, 2B, 3A, 3B and 3C, which have a repeatable motif
as disclosed herein above are referred to herein as "homologous
motif binding peptides". Homologous motif binding peptides will
include 6-25 amino acid residues, preferably 6-15 amino acid
residues and more preferably 6 to 12 amino acid residues. Further a
homologous motif binding peptide will bind to a target with a
binding affinity similar to or greater than the binding affinity to
the same target as a binding peptide of FIG. 1A, 1B, 2A, 2B, 3A, 3B
or 3C having the same repeatable motif. Preferably the target will
be a tannin, phenolic or anthocyanin compound, most preferably a
tea or wine stain, and the binding affinity will be at least about
10.sup.-2M, at least about 10.sup.-3M, at least about 10.sup.-4M,
at least about 10.sup.-6M and generally between about 10.sup.-2M
and 10.sup.-9M.
[0063] A homologous motif binding peptide will include not only a
repeatable motif as defined herein, but also will have between 20%
and 95% amino acid sequence identity with a sequence illustrated in
FIGS. 1A, 1B, 2A, 2B, 3A, 3B and 3C having the same repeatable
motif, that is at least 25% sequence identity, at least 30%
sequence identity, at least 40% sequence, at least 50% sequence
identity, at least 60% sequence identity, at least 70% sequence
identity, at least 80% sequence identity, at least 85% sequence
identity, at least 90% sequence identity or at least 95% sequence
identity to a binding peptide illustrated in FIGS. 1A, 1B, 2A, 2B,
3A, 3B and 3C which includes the same repeatable motif. Preferably
if the homologous motif binding peptide is a 7 amino acid residue
peptide, the homologous motif binding peptide will have at least
30% sequence identity with a binding peptide illustrated in FIGS.
1A, 1B, 2A, 2B, 3A, 3B and 3C having the same repeatable motif when
the peptides are aligned with no gaps. If the homologous motif
binding peptide is a 12 amino acid residue peptide, the peptide
will have at least 25% sequence identity with a binding peptide
illustrated in FIGS. 1A, 1B, 2A, 2B, 3A, 3B and 3C having the same
repeatable motif when the peptides are aligned with no gaps.
C. Polynucleotides Encoding the Binding Peptides
[0064] The present invention encompasses polynucleotides which
encode binding peptides according to the invention. Specifically
polynucleotides include nucleic acid sequences encoding peptides
illustrated in FIGS. 1A, 1B, 2A, 2B, 3A, 3B and 3C (SEQ ID NOs.
1-316) and their C-C derivatives. Additionally, polynucleotides of
the invention will encode binding peptides having at least 70%
amino acid sequence identity to a peptide illustrated in FIG. 1A,
1B, 2A, 2B, 3A, 3B or 3C (SEQ ID NOs. 1-316), their C-C derivatives
and homologous motif binding peptides. As will be understood by the
skilled artisan, due to the degeneracy of the genetic code, a
variety of polynucleotides can encode a binding peptide of the
invention. The present invention encompasses all such
polynucleotides. A polynucleotide which encodes a binding peptide
of the invention may be obtained by standard procedures known in
the art, for example, by chemical synthesis, by PCR and by direct
isolation and amplification.
D. Conjugation of Binding Peptides to an Agent.
[0065] In one embodiment, a binding peptide conjugate is formed
wherein a binding peptide according to the invention is linked with
an agent. While agents may include proteins, carbohydrates, lipids
and ions as described above, various preferred agents are listed
below.
[0066] In one aspect an agent may be a protein. The protein may be
an enzyme, a hormone, a growth factor, a cytokine, an antibody, and
an anti-astringent protein or other protein.
[0067] Enzymes include but are not limited to amylolytic enzymes,
proteolytic enzymes, cellulolytic enzymes, redox enzymes,
transferases and cell wall degrading enzymes. Examples of these
enzymes include, but are not limited to, amylases, proteases,
xylanases, lipases, laccases, phenol oxidases, oxidases, such as
glucose oxidases and galactoses, oxidases, peroxidases, cutinases,
catalases, cellulases, hemicellulases, esterases, pectinases,
glycosidases, isomerases, transferases, galactosidases,
pullulanases, epimerases, phytases, hydroxylases, epoxydases,
alkyltransferases and chitinases.
[0068] Hormones include, but are not limited to,
follicle-stimulating hormone, luteinizing hormone,
corticotropin-releasing factor, somatostatin, gonadotropin hormone,
vasopressin, oxytocin, erthropoietin, insulin and the like.
[0069] Growth factors are proteins that bind to receptors on the
cell surface, with the primary result of activating cellular
proliferation and/or differentiation. Growth factors include, but
are not limited to, platelet-derived growth factors, epidermal
growth factor, nerve growth factor, fibroblast growth factors,
insulin-like growth factors, transforming growth factors and the
like.
[0070] Cytokines are a unique family of growth factors. Secreted
primarily from leukocytes, cytokines stimulate both the humoral and
cellular immune responses, as well as the activation of phagocytic
cells. Cytokines include, but are not limited to, colony
stimulating factors, the interleukins (IL-1 (.alpha.and .beta.),
IL-2 through IL-13) and the interferons (.alpha., .beta. and
.gamma.).
[0071] Antibodies include, but are not limited to, immunoglobulins
from any species from which it is desirable to produce large
quantities. It is especially preferred that the antibodies are
human antibodies. Immunoglobulins may be from any class, i.e., G,
A, M, E or D.
[0072] Anti-astringency compounds include proteins or carbohydrates
that reduce the astringency of other compounds by either binding to
them and/or precipitating them. Anti-astringency proteins include
but are not limited to casein and albumin.
[0073] The agent may also be a vitamin, such as thiamin,
riboflavin, niacin, pantothenic acid, pyridoxal, pyridoxamine,
pyridoxine, biotin, cobalamin, folic acid, ascorbic acid, vitamin
A, vitamin D, vitamin E and vitamin K.
[0074] Sweeteners which may be agents include carbohydrates and
sugar alcohols such as, raw sugar, corn sweetener, corn syrup,
dextrose, sucrose, granulated sugar, brown sugar, confectioner's
sugar, honey, lactose, maltose, mannitol, sorbitol, aspartame.
[0075] An agent may be a bleaching compound, such as an oxygen
bleaching agent or halogen bleaching agent. Non-limiting examples
of oxygen bleaching agents include perborate, percarbonate,
sulfate/hydrogen peroxide, and percarboxylic acid. Non-limiting
examples of halogen bleaching agents include hypohalite and
hypochlorite bleaching agents such as trichloro-isocyanuric acid,
sodium and potassium dichloro-isocyanurate and N-chloro and N-bromo
alkanesulphonamides. Ions such as salts for example potassium,
calcium and bicarbonates may also be agents.
[0076] An agent may also be a dye such as a fluorescent dye. For
example, fluorescein isothiocynate (FITC), rhodamine,
phycoerytherin, phycocyanin, fluorescamine and green fluorescent
protein (GFP). Fluorescent dyes are disclosed in British Patent
Appl. No. 2094826.
[0077] Particularly preferred agents are proteins such as enzymes.
In one embodiment preferred enzymes are oxidoreductase enzymes.
These enzymes include dehydrogenases, reductases, oxidases,
synthases, monooxygenases, isomerases, lipoxygenases, dioxygenases
and hydroxylases. More specifically preferred oxidoreductase
enzymes as agents include laccases (EC 1.10.3.2), phenol oxidases
(EC 1.14.18.1), catalases (EC 1.11.1.6), bilirubin oxidases (EC
1.3.3.5), catechol oxidases (EC 1.10.3.1), peroxidases (EC
1.11.1.7), and glucose oxidases (EC 1.1.3.4). Other preferred
enzymes include amylases, proteases, xylanases, lipases,
transferases and cellulases.
[0078] Numerous references are available on suitable enzymes which
may be linked with a binding peptide according to the invention to
form a binding peptide conjugate. Proteins conjugated with a
binding peptide of the invention may be recombinant proteins or
naturally occurring proteins. Oxidoreductase enzymes, such as
phenol oxidizing enzymes and particularly laccases, and
polynucleotides encoding said enzymes which may be conjugated with
a binding peptide of the invention are disclosed for example in WO
98/27197; WO 98/27198; WO 98/38286; WO 99/49020; WO 00/37654; WO
01/21809; U.S. Pat. No. 4,760,025; U.S. Pat. No. 5,770,419; U.S.
Pat. No. 5,985,818; U.S. Pat. No. 6,060,442; and U.S. Pat. No.
6,168,936. Proteases, such as subtilisins are disclosed in U.S.
Pat. No. 6,197,567; U.S. Pat. No. 6,190,900; U.S. Pat. No.
6,110,884; EP 130756; EP 251446; EP 260105; EP525610; WO 87/04461
and WO 94/02618. Cellulases are disclosed in U.S. Pat. No.
5,989,899; U.S. Pat. No. 6,063,611; U.S. Pat. No. 6,268,328; U.S.
Pat. No. 6,287,839 and U.S. Pat. No. 6,423,524. Amylases are
disclosed in U.S. Pat. No. 6,440,716. Lipases are disclosed in U.S.
Pat. No. 6,156,552; EP 407225; WO 95/06720; WO 95/22615; and WO
96/27002.
[0079] A binding peptide of the invention may act to deliver an
agent to a target. The term deliver or delivering means to assist
in the movement of the agent. In one embodiment the agent,
particularly an enzyme, is delivered to a compound selected from
tannin, polyphenolic or anthocyanin compounds and most particularly
a tannin, polyphenolic or anthocyanin stain on a fabric or
surface.
E. Making the Binding Peptide Conjugate.
[0080] The binding peptide conjugate may be constructed by methods
well known in the art including use of PCR. A binding peptide
according to the invention may be inserted into an agent or
attached to a terminus of the agent. When the agent is a protein a)
the binding peptide may be inserted into the protein b) the binding
peptide may replace an internal loop or turn, and/or c) the binding
agent may be attached to the carbon or nitrogen terminus of the
enzyme. In a preferred embodiment the agent is a protein
(particularly an enzyme) and the binding peptide is linked to the
carbon terminus of the agent. An agent may also be linked to a
binding protein by chemical modification such as by an ester
linkage or an amide linkage. Various methods of conjugating
peptides to an agent are disclosed for example in U.S. Pat. No.
6,348,317; WO 02/57299; WO 02/55543; WO 02/26782; WO 00/48464; and
WO 98/34956.
F. Expression Systems, Transformation and Cultivation of Host
Cells.
[0081] The present invention provides vectors, host cells,
expression methods and systems for the production of the binding
peptides and binding peptide conjugates in host microorganisms,
such as bacteria, fungus and yeast.
[0082] Molecular biology techniques are disclosed in Sambrook et
al., MOLECULAR BIOLOGY CLONING: A LABORATORY MANUAL, 2nd Ed (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. A
polynucleotide encoding a binding peptide or a binding peptide
conjugate is obtained and transformed into a host cell using
appropriate vectors. A variety of vectors and transformation and
expression cassettes suitable for the cloning, transformation and
expression of proteins in fungus, yeast and bacteria are known by
those of skill in the art.
[0083] Vectors will further include initiation control regions or
promoters, which are useful to drive expression of the binding
peptide or binding peptide conjugates in a host cell. Regulatory
control elements are known to those skilled in the art. Virtually
any promoter capable of driving the expression of the particular
agent is suitable for the present invention. Once suitable
cassettes are constructed they are used to transform a host
cell.
[0084] Preferably a host cell is a microbial host cell, and
preferably a bacteria, fungal or yeast host cell. In one embodiment
the host cell is a gram positive bacteria, preferably a Bacillus
species, such as B. subtilis. In another embodiment the host cell
is a gram negative host cell, preferably an Escherichia species,
such as E. coli. In other embodiments the host cell is fungal host
cell, such as a filamentous fungus including a Aspergillus species,
a Trichoderma species and a Mucor species. Particularly preferred
are T. reesei, A. niger and A. oryzae.
[0085] One skilled in the art is well aware of methods of
transforming host cells with polynucleotides encoding a protein of
interest. General transformation procedures are taught in CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (vol. 1, edited by Ausubel et al.,
John Wiley & Sons, Inc. 1987, Chapter 9) and include calcium
phosphate methods, transformation using PEG and electroporation.
For Aspergillus and Trichoderma, PEG and calcium mediated
protoplast transformation can be used (Finkelstein, DB 1992
TRANSFORMATION. IN BIOTECHNOLOGY OF FILAMENTOUS FUNGI. TECHNOLOGY
AND PRODUCTS (eds. by Finkelstein & Bill) 113-156.
Electroporation of protoplast is disclosed in Finkelestein, D B
1992 Transformation. In BIOTECHNOLOGY OF FILAMENTOUS FUNGI.
TECHNOLOGY AND PRODUCTS (eds. by Finkelstein & Bill) 113-156.
Microprojection bombardment on conidia is described in Fungaro et
al. (1995) Transformation of Aspergillus nidulans by
microprojection bombardment on intact conidia. FEMS Microbiology
Letters 125 293-298. Agrobacterium mediated transformation is
disclosed in Groot et al. (1998) Agrobacterium tumefaciens-mediated
transformation of filamentous fungi. Nature Biotechnology 16
839-842. For transformation of Saccharomyces, lithium acetate
mediated transformation and PEG and calcium mediated protoplast
transformation as well as electroporation techniques are known by
those of skill in the art.
[0086] Transformation of Bacillus is described, for example in
Chang and Cohen (1979) Mol. Gen Genet. 168:111-115; Smith et al.
(1986) Appl. and Env. Microbiol. 51:634; Mann et al. (1986) Current
Microbiol. 13: 131-135. Also general reference is made to MOLECULAR
BIOLOGICAL METHODS FOR BACILLUS. Eds. Hardwood and Cutting, John
Wiley & Sons (1990).
[0087] A binding peptide or a binding peptide conjugate,
particularly an enzyme conjugate wherein the enzyme is an
oxidoreductase, a protease, an amylase, a xylanase, a lipase or a
cellulase, may be produced by cultivation of a host cell which
includes a polynucleotide encoding the binding peptide or enzyme
conjugate, under aerobic conditions in nutrient media containing
assimilable carbon and nitrogen together with other essential
nutrient. These conditions are well known in the art.
[0088] Host cells that comprise a coding sequence for a binding
peptide or binding peptide conjugate and express the binding
peptide may be identified by a variety of procedures known to those
of skill in the art. These procedures include, but are not limited
to, DNA-DNA or DNA-RNA hybridization and protein bioassay or
immunoassay techniques which include membrane-based,
solution-based, or chip-based technologies for the detection and/or
quantification of the nucleic acid or protein.
[0089] Once a binding peptide conjugate is encoded the enzyme
conjugate may be isolated and purified from the host cell by
well-known techniques such as, cell separation and concentration of
the cell free broth by ultrafiltration, ammonium sulfate
fractionation, purification by gel filtration, ion exchange or
hydrophobic interaction chromatography, PEG extraction and
crystallization.
[0090] Methods of purification are well-known for many enzymes. One
non-limiting example of purification of an enzyme conjugated to a
binding peptide of the invention includes small-scale purification
(e.g., less than 1 g) of the enzyme using hydrophobic interaction
chromatography. Samples may be filtered and loaded onto a column
containing 20HP2 resin (Perceptives Biosystems), hooked up to a
BioCad workstation (Perceptives Biosystems). The column may be
washed with ammonium sulfate in buffer. Elution of the derivatized
phenol oxidizing enzyme activity can be performed using a salt
gradient ranging from 35% to 0% of a 3M ammonium sulfate solution
in 30 mM Mes Bis Tris Propane buffer at pH 5.4. The fractions
enriched in the derivatized phenol oxidizing enzyme activity can be
monitored using UV absorbance at 280nm and a qualitative ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) activity assay.
The samples can be pooled, concentrated and diafiltered against
water. Enzyme samples purified according to this method are
estimated to be at least about 70% pure.
G. Applications
[0091] The binding peptides and binding peptide conjugates
according to the invention may be used in numerous applications
which include but are not limited to enzyme and cleaning
compositions, food industry applications and personal care
applications. Some of these applications are discussed below, but
the specific examples should not be interpreted as limiting any
general application.
Enzyme and Detergent Compositions.
[0092] A binding peptide conjugate of the present invention may be
used to produce, for example, enzymatic compositions for use in
detergent or cleaning compositions; such as for removing food
stains on fabrics or removing food stains on surfaces such as
ceramic and teeth.
[0093] Enzymatic compositions may also comprise additional
components, such as for example, for formulation or as performance
enhancers. For example, detergent compositions may comprise, in
addition to the binding peptide conjugate, conventional detergent
ingredients such as surfactants, builders and enzymes. Surfactants
include nonionic, anionic and cationic surfactants (see
EP-A-346995). Enzymes include for example, proteases, amylases,
lipases, cutinases, cellulases and peroxidases (U.S. Pat. No.
4,689,297). Other ingredients include enhancers, stabilizing
agents, bactericides, optical brighteners and perfumes. The
enzymatic compositions may take any suitable physical form, such as
a powder, an aqueous or non-aqueous liquid, a paste or a gel.
Reference is made to U.S. Pat. No. 3,929,678; U.S. Pat. No.
4,760,025; U.S. Pat. No.5,011,681; WO 97/04079; WO 97/076202; WO
96/06930; WO 95/01426 and McCutheon's Detergents and Emulsifiers,
North American Ed. (1986) Allured Publishing Co.
[0094] A binding peptide conjugate, particularly when the agent is
an enzyme and more particularly when the agent is a redox enzyme
such as a laccase, can act to modify the color associated with dyes
or colored compounds in the presence or absence of enhancers
depending upon the characteristics of the colored compound. If a
compound is able to act as a direct substrate for the binding
peptide conjugate, the phenol oxidizing enzyme will modify the
color associated with a dye or colored compound in the absence of
an enhancer, although an enhancer may still be preferred for
optimum phenol oxidizing enzyme activity. For other colored
compounds unable to act as a direct substrate for the binding
peptide conjugate or not directly accessible to the conjugate, an
enhancer may be required for optimum enzyme activity and
modification of the color.
[0095] Enhancers are described in for example WO 95/01426, WO
96/06930, and WO 97/11217. Enhancers include but are not limited to
phenothiazine-10-propionic acid (PTP), 10-methylphenothiazine
(MPT), phenoxazine-10-propionic acid (PPO), 10-methylphenoxazine
(MPO), 10-ethylphenothiazine-4-carboxylic acid (EPC)
acetosyringone, syringaldehyde, methylsyringate,
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS), 2,6
dimethoxyphenol (2,6-DMP), and guaiacol(2-methoxyphenol).
[0096] While enzymes and their use in detergent and cleaning
compositions are well known, a main advantage of a binding peptide
conjugate according to the invention is the delivery of an agent to
a target and the enhanced binding of the conjugate to a target
stain compared to the agent without the binding peptide.
Food Industry Applications.
[0097] Tannins are important taste components in tea and wine. In
wine, tannins come from the skin and seed of red grapes and from
the wooden oak barrels used in the fermentation and aging process.
Tannins, which can bind to proteins in saliva, cause the proteins
to precipitate and result in a stringent or bitter taste. Various
tannins are found in wine during the early stages of the
fermentation process. During the later stages of the fermentation
process many of these tannins are extracted from the wine. A
binding peptide or a binding peptide conjugate according to the
invention may be particularly useful in this wine aging process. By
targeting a tannin compound in the early stage of the wine
fermentation process the astringency of tannins could be reduced or
eliminated in the wine.
Personal Care Applications.
[0098] Tannins and anthocyanin compounds are natural dyes and may
act as ultraviolet light protectants, tan enhancers and
astringents. By either direct addition of tannin or anthocyanin
binding peptides, which may displace tannins or anthocyanins from
the compounds they bind, or by addition of a binding peptide
conjugate one could modify the action of these compounds on various
biological tissues particularly teeth, nails and skin. A
non-limiting example includes a conjugated binding peptide
comprising a peptide linked to a bleaching agent, wherein the
conjugate delivers the bleaching agent to stained teeth for the
purpose of bleaching. Another non-limiting example includes
providing a binding peptide to the skin wherein astringency may be
modified. Personal care products including a binding peptide may be
formulated as creams, lotions, ointments and the like.
[0099] Having thus described the binding peptides and binding
peptide conjugates of the present invention, the following examples
are now presented for the purposes of illustration and are neither
meant to be, nor should they be, read as being restrictive.
Dilutions, quantities, etc. which are expressed herein in terms of
percentages are, unless otherwise specified, percentages given in
terms of percent weight per volume (w/v). As used herein,
dilutions, quantities, etc., which are expressed in terms of %
(v/v), refer to percentage in terms of volume per volume.
Temperatures referred to herein are given in degrees centigrade
(C).
Experimental
EXAMPLE 1
Selection of the Binding Peptides on Tea and Wine Stained
Cotton
[0100] While a number of selection techniques may be used to screen
for binding peptides, the majority of the binding peptides
according to the invention were selected according to the method
described herein below.
[0101] 10 microliters of a commercially (New England Biolabs)
available phage display library either a cyclic 7-mer (at
2.times.10.sup.3 pfu/ml) or a linear 12-mer (at 4.times.10.sup.12
pfu/ml) were pre-incubated with a cotton swatch in a pre-blocked
and washed 96 well plate in the presence of a 150 .mu.l
Tris-buffered saline (TBS) solution (at 2.times.10.sup.-5 g/l for
the cyclic 7-mer, 2.times.10.sup.-3g/l for the linear 12-mer) of
detergent, pH 10 for 20 minutes using gentle shaking. The solution
was pipetted off and added to a second cotton swatch for 20 minutes
under gentle shaking. This process was repeated a third time. The
solution was pipetted off and added to a tea or wine stained cotton
swatch (Textile Innovators Corp. Windsor, N.C.) for 60 minutes
under gentle agitation. The solution was drawn off and discarded.
The stained swatch was washed 5 times for 5 minutes each with 200
.mu.l of TBST (TBS containing 0.1% Tween 20). The swatch was
transferred to an empty well using sterile tips, washed as
described above, and transferred to another empty well. 15 .mu.l of
a glycine 0.2M solution pH 2.2 was added to the stained swatch and
the plate was shaken for less than 10 minutes. This solution was
neutralized by the addition of 100 .mu.l of a Tris HCL 1M solution,
pH 9.1 for 10 minutes. The solution, which constitutes the acid
eluted peptide population, was pipetted off and stored at 4.degree.
C. until further use.
EXAMPLE 2
Amplification of the Acid Eluted Peptides
[0102] 4.times.20 .mu.l of the acid eluted phage peptide population
was used to infect 4.times.400 .mu.l E. coli (New England BioLabs)
grown to an OD at 610 nm of 0.3 to 0.65 from a 100.times. dilution
in LB of an overnight culture. The cells were plated on 4.times.140
mm LB plates in the presence of IPTG (Sigma) (40 .mu.l at 20 mg/ml
per plate) and Xgal (Sigma) (40 .mu.l at 40 mg/ml of DMF per
plate), added to 5 mls of melted top agarose, and left to incubate
overnight at 37.degree. C. The 4 plates were scraped with a sterile
glass microscope slide and the scrapings were pushed through an
18.5 gage needle of a 60 ml syringe into a sterile conical tube; 50
ml of TBS was added to the tube and the capped tube was left to
shake on a rocker at room temperature for at least 14 hrs. The
contents of the tube were centrifuged at 10,000 rpm for 30 minutes
in sterile Oakridge tubes at 4.degree. C. The supernatant was
collected and the phage precipitated by adding 1/6 volume of a 20%
polyethylene glycol (PEG)/2.5 M NaCl solution. This was left to
incubate at 4.degree. C. for at least 4 hours and preferably
overnight. The solution was then spun at 10,000 rpm for 30 minutes
at 4.degree. C. and the supernatant discarded. The pellet was
resuspended in 1 ml of TBS and transferred to a sterile Eppendorff
tube. The phage was reprecipitated with 1/6 volume of a 20% PEG/2.5
M NaCl solution with incubation on ice for at least 1 hour. This
was followed by another centrifugation at 10,000 rpm for 10 min at
4.degree. C. The supernatant was discarded, the tube re-spun
briefly, and residual supernatant removed. The pellet was
resuspended in 200 .mu.l TBS/0.02% NaN.sub.3, spun to remove
insoluble material and transferred.
EXAMPLE 3
Biopanning
[0103] The amplified phage peptide populations from the first round
of deselection on cotton/selection of stained cotton swatches were
submitted to another round of deselection and selection as
described above. For the cyclic 7-mer peptide library
2.times.10.sup.-4 g/l TBS was used, and for the linear 12-mer
peptide library 2.times.10.sup.-2 g/l TBS was used. After acid
elution and amplification of the phage, a third round of biopanning
was performed. The third round used 2.times.10.sup.-3 g/l TBS of
detergent for the cyclic 7-mer phage peptides and 2.times.10.sup.-1
g/l TBS for the linear 12-mer phage peptides. After acid elution
and amplification, a fourth round of biopanning was used and 2 g/l
of detergent dissolved in water in one experiment and TBS in
another were used for both types of phage peptides. The phage
peptides were acid eluted and amplified from the fourth round of
biopanning and selected in a fifth round of biopanning wherein the
Tween 20 concentration was increased from 0.1% to 0.8% in the wash
conditions. Additionally a round of selection on tea and wine was
performed using the phage peptides from the third round as
described above. In this fourth round of biopanning, 2 g/l of
detergent in water in the wash conditions was used. One skilled in
the art is well aware that various parameters as described
hereinabove may be varied without affecting the nature of the
invention. The above described method is one method which may be
used to screen for binding peptides of the invention.
EXAMPLE 4
Selection of the Binding Peptides on Stained Cotton After
Biopanning
[0104] 225 .mu.l of a 1/100 dilution of an overnight culture of E.
coli cells in LB broth were incubated with phage plaques using
sterile toothpicks in a sterile 96-well V-bottom plate. A replica
plate was made for glycerol stocks of the phage peptides. The
plates were covered with porous Qiagen plate sealers and shaken for
4 hours at 37.degree. C. at 280 rpm in a humidified shaker box and
then spun at 4000 rpm for 30 min at 4.degree. C. 160 .mu.l of the
phage peptides supernatant was transferred to another 96-well
V-bottom plate containing 64 .mu.l of 20% PEG/2.5 M NaCl. The
plates were left to shake for 5 minutes and then left to stand for
10 minutes. The glycerol stock plate was prepared by adding 100
.mu.l phage supernatant to 150 .mu.l 75% glycerol solution in a
sterile 96 well plate which was then sealed with parafilm, labeled,
and stored at -70.degree. C. until further use.
[0105] The PEG precipitated phage plate was centrifuged at 4000 rpm
for 20 minutes at 4.degree. C. The plate was inverted rapidly to
remove excess PEG/NaCl and left upside down on a clean paper towel
to drain residual fluid. 60 .mu.l of iodide salt solution (10 mM
Tris.HCl, pH 8.0, 1 mM EDTA, 4 M NaI) were added to each well and
the phage pellets thoroughly resuspended by shaking the plate
vigorously for 5 minutes. 150 .mu.l of 100% EtOH were added and the
plate was spun at 4000 rpm for 20 minutes at 4.degree. C., the
supernatants discarded and the plate blotted. The pellets were
washed with 225 .mu.l of 70% EtOH without disturbing the pellets;
the plate was inverted and left to air-dry for at least 30 minutes.
The pellets were resuspended in 30 .mu.l of Tris.HCl 10 mM, pH 8.5
buffer by shaking the plate for 30 minutes at full speed. 1 .mu.l
of g96 reverse primer (obtained from New England BioLabs, 3.4 pmole
per tube) was added to 11 .mu.l of DNA pellet sample and the
contents submitted for sequencing on a ABI Applied Biosystem
373XL.
[0106] By raising the concentration of detergent in every round of
biopanning and additionally during the washes, the stringency of
the selection and wash steps was increased. In so doing, only those
peptides that bind specifically to compounds in tea or wine remain
bound after successive selection/wash steps in increasing detergent
concentrations. Accordingly, increasing concentration of detergent
between biopanning rounds, improves the number of phage that
contains real peptide binders, and reduces the number of
false-positives. Thus this approach helps improve the signal to
noise ratio in this biopanning procedure.
[0107] FIGS. 1A-1B and 3A-3C (SEQ ID NOs.1-111 and 206-316)
illustrate the amino acid sequences of numerous binding peptides
determined according to the method described in examples 1-4.
EXAMPLE 5
Selection of the Phage Binding Peptides on Tea Stained Ceramic
[0108] Deselection as described above was performed three times on
unstained pieces of a ceramic teapot in a blocked 96 well plate
using either cyclic 7-mer, linear 7-mer, and or linear 12-mer phage
peptide libraries in the presence of a commercially available dish
detergent. Selection was then performed on tea stained pieces of
ceramic. The tea stained ceramic pieces were rinsed in TBST. After
acid elution and neutralization, the tea stained ceramic pieces
were further rinsed in TBS, dried and placed in PCR tubes. Lysis
buffer was added to the tea stained ceramic and lysis was
performed. The lysis solutions were subjected to a series of PCR
reactions, TA cloning and sequencing as described above. The
peptides were analyzed for the presence of repeatable motifs.
Additionally, the PCR products in the TA cloning step were
amplified using PCR. The PCR fragments were digested with
restriction enzymes and the resulting fragments were purified using
standard phenol/chloroform extraction and ethanol precipitation
procedures. The fragments were eluted on a 8% PA gel in TBE and
fragments of interest were cut out with a razor blade and further
purified using the Qiagen purification kit procedure. The purified
fragments were ligated with vector and competent E. coli E2537
cells were transformed using well known techniques. The
transformants were sequenced according to standard protocols and
the corresponding phage peptide libraries amplified prior to a
second round of selection and deselection.
[0109] Using amplified phage peptide libraries from the first round
of selection, another round of deselection and selection was
performed as described above. The tea stained ceramic pieces were
rinsed with TBST prior to acid elution and neutralization. The
pieces were then rinsed in TBS and dried. The phage peptide
libraries bound to the tea stained ceramic were lyzed and their DNA
amplified using a series of PCR reactions. TA cloning was preformed
on the PCR products. The TA clones were picked for PCR and
sequenced as described previously herein. The sequences were also
analyzed for the presence of repeatable motifs.
[0110] FIGS. 2A-2B (SEQ ID NOs. 112-201) illustrate the amino acid
sequence of numerous binding peptides determined according to the
method described in this example 5.
EXAMPLE 6
Selective Binding of Phage Bound Peptides to Tea Stained Cotton
Swatches
[0111] The phages containing the peptides LHQNQKS (SEQ ID NO. 68),
TNNTSPT (SEQ ID NO. 24), SWNTSPL (SEQ ID NO. 80), SYGPMTN (SEQ ID
NO. 65), PNTTRHS (SEQ ID NO. 2), LWTSPQL (SEQ ID NO. 8), and WT
phage (without a peptide insert) were amplified from the glycerol
stocks described in example 4. The amplification procedure was done
as described as in example 2. A drop of corresponding glycerol
stock was added to 20 ml of LB broth containing 0.2 ml of an
overnight culture of E. coli. The culture was left to grow at
37.degree. C. under vigorous shaking for 4.5 hrs, transferred to
sterile 50 ml Oakridge tubes and centrifuged at 10 000 rpm for 10
min. The supernatants (17 mls) were added to fresh, sterile
centrifuge tubes containing 3 ml of 20% PEG/2.5M NaCl solution (1/6
volume). The phages were precipitated at 4.degree. C. for at least
4 hr and then centrifuged for 15 min at 10,000 rpm at 4.degree. C.
The phage pellets were suspended in 1 ml of TBS, transferred to
sterile 1.5 ml microfuge tubes and re-precipitated with 1/6 volume
PEG/NaCl on ice for at least 1 hr. The tubes were again spun for 10
min at 4.degree. C. and supernatants discarded. The pellets were
suspended in 0.2 ml of TBS/0.02% NaN.sub.3 solution and the
solutions were spun for 1 min to remove any insoluble material.
Supernatants were transferred to sterile screw cap tubes.
[0112] Small (1/8'') swatches of tea stained cotton (Textile
Innovators Corp. Windsor, N.C.) and unstained cotton were punched
out (in duplicates for each phage peptide) and placed in a
pre-blocked and washed multititer plate. 0.150 ml of a 10.times.
dilution of each titered phage peptide solution in detergent/TBS
(0.001 g of detergent/L of TBS) was added to two tea stained
swatches and to two unstained cotton swatches and left to incubate
for 30 min at room temperature on a rocker, under mild agitation.
Solution was pipetted off and the swatches were rinsed 9 times with
0.2 ml of a TBST solution. Swatches were transferred into fresh
empty wells and rinsed another 9 times with 0.2 ml of a TBS
solution. Each swatch was placed in a PCR tube. 0.1 ml of lysis
buffer was added (10 mM Tris.HCl, pH8.4, 0.1% Triton X100), and
then subjected to lysis at 95.degree. C. for 10 min. 2 .mu.l of a
100.times. dilution in lysis buffer (of the contents of each PCR
tube) were added to Light Cycler.TM. (Roche) capillaries. 10 .mu.l
of the Light Cycler.TM. cocktail (Roche. Per tube: 5.3 ul water,
1.1 ul of MgCl.sub.2, 1.2 .mu.l of mix (ATP.sub.+ dye), 2.4 ul of
primers) was added to the tubes and the contents briefly spun on a
table top centrifuge. The capillary tubes were capped and run on
the Light Cycler.TM. PCR instrument. The contents of the tubes were
quantified against a series of dilutions of a known and quantified
phage peptide standard. The fluorescent signal coming from the
intercalating dye correlates to the amount of DNA (copies) (using
melting point correction) and therefore number of phage peptide
present.
[0113] FIG. 4 shows selective binding of phage-bound peptides to
tea stained cotton swatches as compared to non-stained cotton
swatches. For each phage-bound peptide illustrated, the peptide
binds to tea stained cotton at least 2 times greater than to
non-stained cotton. The graph shows phage peptide sequences which
contain repeatable motifs bind greater than WT. LWTSPQL (SEQ ID NO.
8) binds to tea stained cotton about 2.5 times more than WT binds
to tea stained cotton. LWTSPQL (SEQ ID NO. 8) binds to tea stain
about 5 times more than to non-stained cotton. PNTTRHS (SEQ ID NO.
2) binds to tea stain about 15 times more than non-stained cotton.
Sequence CWU 1
1
33217PRTArtificial Sequencebinding peptide 1Lys Thr Pro Ser Pro His
Gly1 527PRTArtificial Sequencebinding peptide 2Pro Asn Thr Thr Arg
His Ser1 537PRTArtificial Sequencebinding peptide 3Gly Lys Met Tyr
Leu Lys Ala1 547PRTArtificial Sequencebinding peptide 4Asn Gln Ser
His Met Pro Arg1 557PRTArtificial Sequencebinding peptide 5Lys Thr
Pro Ser Ser Met Glu1 567PRTArtificial Sequencebinding peptide 6Ile
Glu Lys Pro His Ala Asp1 577PRTArtificial Sequencebinding peptide
7Pro Thr Tyr His His Gln Thr1 587PRTArtificial Sequencebinding
peptide 8Leu Trp Thr Ser Pro Gln Leu1 597PRTArtificial
Sequencebinding peptide 9His Ser Leu Asp Gln Arg Ser1
5107PRTArtificial Sequencebinding peptide 10Gln Ile Gln Thr Pro Leu
Ser1 5117PRTArtificial Sequencebinding peptide 11Ser Leu Asn Asn
Thr Asn Thr1 5127PRTArtificial Sequencebinding peptide 12Val Pro
Thr Gly Lys Leu Gln1 5137PRTArtificial Sequencebinding peptide
13Pro Thr Trp Ser Leu Ala Met1 5147PRTArtificial Sequencebinding
peptide 14Leu Gly Thr Pro Gln Gln Thr1 5157PRTArtificial
Sequencebinding peptide 15Ser Phe Tyr His Gln Thr Tyr1
5167PRTArtificial Sequencebinding peptide 16Met Met Asn Ser Trp His
Lys1 5177PRTArtificial Sequencebinding peptide 17Lys Gln Ser Gln
Pro Gln Thr1 5187PRTArtificial Sequencebinding peptide 18Gln Lys
His Ser Pro Gly His1 5197PRTArtificial Sequencebinding peptide
19Thr Ser Gly Ser Met His Ser1 5207PRTArtificial Sequencebinding
peptide 20Val Leu Arg Pro Ala Ala Leu1 5217PRTArtificial
Sequencebinding peptide 21Ser Pro Met Asp Glu Tyr Tyr1
5227PRTArtificial Sequencebinding peptide 22Leu Lys Ser Thr Arg Pro
Thr1 5237PRTArtificial Sequencebinding peptide 23Asp Ser Arg Leu
Ser Arg Ser1 5247PRTArtificial Sequencebinding peptide 24Thr Asn
Asn Thr Ser Pro Thr1 5257PRTArtificial Sequencebinding peptide
25Gln Thr Gln Pro Pro Gly Ser1 5267PRTArtificial Sequencebinding
peptide 26Leu Lys Asn Val Pro Phe Arg1 5277PRTArtificial
Sequencebinding peptide 27His Asn Tyr Thr Leu Gln Lys1
5287PRTArtificial Sequencebinding peptide 28Lys Phe Met His Thr Thr
Ala1 5297PRTArtificial Sequencebinding peptide 29Lys Thr Ser Ile
Gly His Trp1 5307PRTArtificial Sequencebinding peptide 30Leu Pro
Ala Lys Pro Leu Ala1 5317PRTArtificial Sequencebinding peptide
31Leu Asn Ser Ser Ser Lys Ser1 5327PRTArtificial Sequencebinding
peptide 32Asn Lys Ser Asn Ala Thr Val1 5337PRTArtificial
Sequencebinding peptide 33Ala Leu Asn Pro Leu His Thr1
5347PRTArtificial Sequencebinding peptide 34Asp Thr His Thr Gln Lys
His1 5357PRTArtificial Sequencebinding peptide 35Thr Ala Gln Ile
His Ser Val1 5367PRTArtificial Sequencebinding peptide 36Thr Met
Ala Pro Ala Lys Asn1 5377PRTArtificial Sequencebinding peptide
37Ser His Leu Asp Lys Arg Leu1 5387PRTArtificial Sequencebinding
peptide 38Thr Asn Trp Asp Ala Pro Thr1 5397PRTArtificial
Sequencebinding peptide 39Thr Met Ser Arg Val Asn Gln1
5407PRTArtificial Sequencebinding peptide 40Thr Thr Thr Asn Lys Pro
Leu1 5417PRTArtificial Sequencebinding peptide 41Leu Thr Gln Asn
Phe His Ser1 5427PRTArtificial Sequencebinding peptide 42Asn Pro
Thr Gln Leu Gln Asn1 5437PRTArtificial Sequencebinding peptide
43Ser Pro Thr Ser Thr Asn Ser1 5447PRTArtificial Sequencebinding
peptide 44Pro Gly Ser Asn Ala Thr Gln1 5457PRTArtificial
Sequencebinding peptide 45Ser Gln Asp Thr Pro Met Tyr1
5467PRTArtificial Sequencebinding peptide 46Thr Asp Pro Ser Met Met
Asn1 5477PRTArtificial Sequencebinding peptide 47Gly Gln Ala Asp
Arg Leu Gln1 5487PRTArtificial Sequencebinding peptide 48Thr Pro
Gln Arg Leu Leu Thr1 5497PRTArtificial Sequencebinding peptide
49Gln Trp Thr Asp Pro Met Lys1 5507PRTArtificial Sequencebinding
peptide 50Ala Lys Thr Asn Leu Lys Asp1 5517PRTArtificial
Sequencebinding peptide 51Glu Val Thr Thr Val Arg Asn1
5527PRTArtificial Sequencebinding peptide 52Leu Asn Asn Tyr Arg Asn
Met1 5537PRTArtificial Sequencebinding peptide 53Ser Gln Met Ser
Pro Leu His1 5547PRTArtificial Sequencebinding peptide 54Ser Ala
Arg Ser Leu Pro Phe1 5557PRTArtificial Sequencebinding peptide
55Pro Thr His Leu Ala Asn Met1 5567PRTArtificial Sequencebinding
peptide 56Thr Gln Lys Asp Asp Arg Gln1 5577PRTArtificial
Sequencebinding peptide 57Thr Asp Phe His Gln Gly Thr1
5587PRTArtificial Sequencebinding peptide 58Thr Tyr Glu Ala Pro Arg
Thr1 5597PRTArtificial Sequencebinding peptide 59Leu Gln Ile Asn
Ala Gln Thr1 5607PRTArtificial Sequencebinding peptide 60His Val
Trp Ser Gln Ala Trp1 5617PRTArtificial Sequencebinding peptide
61His Gln Gly Pro Leu Arg Thr1 5627PRTArtificial Sequencebinding
peptide 62Thr Gln Asn Pro Thr His Ser1 5637PRTArtificial
Sequencebinding peptide 63Glu Asn Ser Phe Ala Gly Gly1
5647PRTArtificial Sequencebinding peptide 64His Gly Ser Ser Ala His
Pro1 5657PRTArtificial Sequencebinding peptide 65Ser Tyr Gly Pro
Met Thr Asn1 5667PRTArtificial Sequencebinding peptide 66Pro Thr
Glu Thr Pro Gln Met1 5677PRTArtificial Sequencebinding peptide
67His Ala Gln Leu Ala Ser Leu1 5687PRTArtificial Sequencebinding
peptide 68Leu His Gln Asn Gln Lys Ser1 5697PRTArtificial
Sequencebinding peptide 69His Pro Ser Asp Thr Ile Leu1
5707PRTArtificial Sequencebinding peptide 70Thr Thr Ala Ala Pro Gln
Met1 5717PRTArtificial Sequencebinding peptide 71Ser Ser Asn Leu
Pro Phe Ala1 5727PRTArtificial Sequencebinding peptide 72Thr Gly
Pro Glu Gln Gly Leu1 5737PRTArtificial Sequencebinding peptide
73Asn Val Ala Ser Thr Arg Ile1 5747PRTArtificial Sequencebinding
peptide 74Ser Asn Val His His Pro Ser1 5757PRTArtificial
Sequencebinding peptide 75Thr Leu Ala His Arg Gly Glu1
5767PRTArtificial Sequencebinding peptide 76Asn Ala Pro His Arg Thr
Leu1 5777PRTArtificial Sequencebinding peptide 77Thr Thr Thr Thr
Pro Phe Ala1 5787PRTArtificial Sequencebinding peptide 78Thr Thr
Gln Met Gln Arg Met1 5797PRTArtificial Sequencebinding peptide
79Ser Thr Met Thr Arg Ala Thr1 5807PRTArtificial Sequencebinding
peptide 80Ser Trp Asn Thr Ser Pro Leu1 5817PRTArtificial
Sequencebinding peptide 81Pro Asp Ser Gln Thr Gly Asn1
5827PRTArtificial Sequencebinding peptide 82Pro Ser Pro Pro Thr Asn
Gln1 5837PRTArtificial Sequencebinding peptide 83Ile Lys Pro Glu
Leu Met His1 5847PRTArtificial Sequencebinding peptide 84Asn Glu
Ile Asn Ala Gln Ser1 5857PRTArtificial Sequencebinding peptide
85Pro Leu Thr Ser Thr Gln Pro1 5867PRTArtificial Sequencebinding
peptide 86Pro Pro Asn Ser Ser Ser Ser1 5877PRTArtificial
Sequencebinding peptide 87His Val Ser Asp Leu Ala Gly1
5887PRTArtificial Sequencebinding peptide 88Thr Leu Ser Arg Thr Thr
Ala1 5897PRTArtificial Sequencebinding peptide 89Ile Asn Lys Thr
Met Ala Glu1 5907PRTArtificial Sequencebinding peptide 90His Leu
Arg Ser Thr Thr Asp1 5917PRTArtificial Sequencebinding peptide
91Asn Ser Pro Asn Pro Ser Ile1 5927PRTArtificial Sequencebinding
peptide 92Thr Gln Met Pro Lys Gln Gln1 5937PRTArtificial
Sequencebinding peptide 93Ser Pro Met Gln Pro Arg Leu1
5947PRTArtificial Sequencebinding peptide 94Phe Thr Ala Asn Leu Arg
Ala1 59512PRTArtificial Sequencebinding peptide 95Phe Asn Ser Met
Ser Asn Ser Arg Gly Tyr Ala Leu1 5 109612PRTArtificial
Sequencebinding peptide 96Leu Phe Leu Pro Pro Thr Pro Pro Pro Glu
Pro Leu1 5 109712PRTArtificial Sequencebinding peptide 97Gln Ala
Val Lys Ala Ser His Ala Thr Met Tyr Leu1 5 109812PRTArtificial
Sequencebinding peptide 98Thr Pro Ala Ser Ile Met Arg Met Pro Leu
Pro Gly1 5 109912PRTArtificial Sequencebinding peptide 99Glu Thr
Gln Pro Ser Ala Met Gly Gly Ser Ser Leu1 5 1010012PRTArtificial
Sequencebinding peptide 100Val Ala Lys Gln Val Thr Thr Pro Gly Ser
Ser Thr1 5 1010112PRTArtificial Sequencebinding peptide 101Thr Tyr
Asn Asp Ile Gln Ala Pro Val Pro Ser Leu1 5 1010212PRTArtificial
Sequencebinding peptide 102Ser Thr Ser Trp Pro Pro Gln Pro His Leu
Ser Pro1 5 1010312PRTArtificial Sequencebinding peptide 103Thr Tyr
Pro Leu Gln Thr Ala Ala Ser Arg Leu Gln1 5 1010412PRTArtificial
Sequencebinding peptide 104Ser Tyr Asp Leu Ile Pro Pro Arg Ser Gly
Leu Ala1 5 1010512PRTArtificial Sequencebinding peptide 105Asn Thr
Thr Gln Thr Leu Arg His Val Ser Leu Ala1 5 1010612PRTArtificial
Sequencebinding peptide 106Thr Ile Val Gly Pro Ile Ile Gly Gly Thr
Ala Gly1 5 1010712PRTArtificial Sequencebinding peptide 107Thr Ser
Gly Phe Asp Arg Ala Leu Ser Pro Ser Leu1 5 1010812PRTArtificial
Sequencebinding peptide 108Asp Leu His Asn His Gln Thr Thr Ser Leu
Arg Tyr1 5 1010911PRTArtificial Sequencebinding peptide 109Arg Xaa
Xaa Val Asp Xaa Pro Pro Pro Ala Leu1 5 1011012PRTArtificial
Sequencebinding peptide 110Ser Ala Leu Glu Gln Ser Thr Glu Arg Pro
Pro Ser1 5 1011111PRTArtificial Sequencebinding peptide 111Ser Asn
Ser Thr Met Asn Ala Leu Ala Pro Ala1 5 101127PRTArtificial
Sequencebinding peptide 112Gln Ser Thr Asp Leu Gln Ala1
51137PRTArtificial Sequencebinding peptide 113Ala Ile Ser Ile Thr
Gly Ser1 511412PRTArtificial Sequencebinding peptide 114Ala Leu Gly
Xaa Ile Pro Xaa Thr Ala His Gln Trp1 5 101157PRTArtificial
Sequencebinding peptide 115Ala Arg Ser Ile Gln Pro Phe1
51167PRTArtificial Sequencebinding peptide 116Ala Thr Val Ile Leu
Thr Asp1 51177PRTArtificial Sequencebinding peptide 117Asp Ala His
Pro Thr Arg Thr1 51187PRTArtificial Sequencebinding peptide 118Asp
Pro Asn Thr Thr Ser His1 51197PRTArtificial Sequencebinding peptide
119Glu Pro Ala Pro Pro Arg Lys1 51207PRTArtificial Sequencebinding
peptide 120Phe Leu Pro Leu Leu Thr Leu1 51217PRTArtificial
Sequencebinding peptide 121Phe Gln Leu Ile Pro Thr Gly1
51227PRTArtificial Sequencebinding peptide 122Gly Ala Phe Phe Thr
Ala Tyr1 51237PRTArtificial Sequencebinding peptide 123Gly His Pro
Gln Leu Pro Leu1 51247PRTArtificial Sequencebinding peptide 124Gly
Pro Ser Xaa Leu Trp Xaa1 51257PRTArtificial Sequencebinding peptide
125Gly Val Pro Phe Ala Thr Pro1 51267PRTArtificial Sequencebinding
peptide 126His Asn Leu Arg Phe Ala His1 51277PRTArtificial
Sequencebinding peptide 127His Arg His Pro Pro Gly Leu1
51287PRTArtificial Sequencebinding peptide 128His Thr Asp Gln Thr
Ser Asp1 512912PRTArtificial Sequencebinding peptide 129His Xaa Gly
Pro Arg Leu Glu Xaa Ala Ser Asp Phe1 5 1013011PRTArtificial
Sequencebinding peptide 130Ile Pro Leu Ile Lys Gly Met His Pro Pro
Asp1 5 101317PRTArtificial Sequencebinding peptide 131Ile Pro Thr
Thr Arg Gln Thr1 51327PRTArtificial Sequencebinding peptide 132Lys
Ala Ser His Leu Val Pro1 51337PRTArtificial Sequencebinding peptide
133Lys Asp Ala Lys Lys Ile Thr1 513412PRTArtificial Sequencebinding
peptide 134Lys Asp Pro Ser Trp Pro Ser Gln Ala Gln Thr Pro1 5
101357PRTArtificial Sequencebinding peptide 135Lys Pro Xaa Leu Pro
Thr Xaa1 51367PRTArtificial Sequencebinding peptide 136Leu Lys Glu
Phe Gln Gln Ile1 51377PRTArtificial Sequencebinding peptide 137Leu
Leu Leu Ser Pro Pro Pro1 51387PRTArtificial Sequencebinding peptide
138Leu Pro Lys His Thr Leu Thr1 51397PRTArtificial Sequencebinding
peptide 139Leu Pro Thr Ser Thr Leu Thr1 51407PRTArtificial
Sequencebinding peptide 140Leu Gln Asp Cys Leu Arg Asn1
51417PRTArtificial Sequencebinding peptide 141Leu Ser Thr Pro Gly
Met Gln1 51427PRTArtificial Sequencebinding peptide 142Leu Thr Pro
Asp Ala Ile Phe1 51437PRTArtificial Sequencebinding peptide 143Met
Val Gln Gly Thr Ser Glu1 51447PRTArtificial Sequencebinding peptide
144Asn Leu Lys Val Gln Gln Arg1 51457PRTArtificial Sequencebinding
peptide 145Asn Ser Ala Pro His Val Thr1 51467PRTArtificial
Sequencebinding peptide 146Asn Thr Asn Pro Phe Gln Pro1
51477PRTArtificial Sequencebinding peptide 147Asn Val Thr Met Val
Leu Leu1 51487PRTArtificial Sequencebinding peptide 148Asn Xaa Lys
Thr Ser Gln Xaa1 51497PRTArtificial Sequencebinding peptide 149Pro
Gly Lys His Gly Gln Ala1 51507PRTArtificial Sequencebinding peptide
150Pro Ile Thr Pro Val Xaa Ala1 51517PRTArtificial Sequencebinding
peptide 151Pro Pro Ile Ile Asp Leu Glu1 51527PRTArtificial
Sequencebinding peptide 152Pro Pro Ser Pro Leu Thr Pro1
51537PRTArtificial Sequencebinding peptide 153Pro Gln Xaa Gly Ile
Xaa Xaa1 51547PRTArtificial Sequencebinding peptide 154Pro Thr Leu
Ala Gly Ala Ser1 51557PRTArtificial Sequencebinding peptide 155Pro
Thr Leu Phe Lys Glu His1 51567PRTArtificial Sequencebinding peptide
156Pro Tyr Leu Ser Asp Lys Ala1 51577PRTArtificial Sequencebinding
peptide 157Gln Asp Thr Ala Pro Leu Thr1 51587PRTArtificial
Sequencebinding peptide 158Gln Asn Gln Lys Ser Thr Thr1
51597PRTArtificial Sequencebinding peptide 159Gln Pro Gly His Leu
Asp Ile1 51607PRTArtificial Sequencebinding peptide 160Gln Ser Asp
Met His Trp Arg1 51617PRTArtificial Sequencebinding peptide 161Gln
Ser Glu Pro His Pro Lys1 51627PRTArtificial Sequencebinding peptide
162Arg Ala Gly Glu Ser His Arg1 51637PRTArtificial Sequencebinding
peptide 163Arg Asp Ala Tyr Leu Thr Pro1 51647PRTArtificial
Sequencebinding peptide 164Arg Leu Ser Leu Pro Met Gln1
51657PRTArtificial Sequencebinding peptide 165Arg Met Ala Thr Pro
Asn Ala1 51667PRTArtificial Sequencebinding peptide 166Ser Ala Ser
Ala Thr Trp Thr1 51677PRTArtificial Sequencebinding peptide 167Ser
Gly Pro Ala Asp Ala Asp1 51687PRTArtificial Sequencebinding peptide
168Ser Ile Ile Pro Pro Arg Gln1 51697PRTArtificial Sequencebinding
peptide 169Ser Lys Asn Thr Ala Phe Gly1 51707PRTArtificial
Sequencebinding peptide 170Ser Leu Ser Thr Xaa Ala Asn1
51717PRTArtificial Sequencebinding peptide 171Ser Met Trp Gly Asn
Leu His1 51727PRTArtificial Sequencebinding peptide 172Ser Asn His
Leu Ile Gln Tyr1 51737PRTArtificial Sequencebinding peptide 173Ser
Arg Ala Trp Ser Trp Pro1 51747PRTArtificial Sequencebinding peptide
174Ser Ser Leu Leu Pro Arg Ser1 51757PRTArtificial Sequencebinding
peptide 175Ser Val Ser Leu Val Ser Leu1 51767PRTArtificial
Sequencebinding peptide 176Ser Xaa Thr Leu Ser Pro Tyr1
51777PRTArtificial Sequencebinding peptide 177Thr Ala Pro Leu Ile
Ser Ile1 51787PRTArtificial Sequencebinding peptide 178Thr Ile Gln
Ser Arg Tyr Thr1 51797PRTArtificial Sequencebinding peptide 179Thr
Lys Ser Ser Met Pro Thr1 51807PRTArtificial Sequencebinding peptide
180Thr Lys Thr Thr Trp Gln Thr1 51817PRTArtificial Sequencebinding
peptide 181Thr Leu Phe Tyr Thr Lys Xaa1 51827PRTArtificial
Sequencebinding peptide 182Thr Gln Arg Leu Thr Thr His1
51837PRTArtificial Sequencebinding peptide 183Thr Arg Glu Ser Gly
Glu Gln1 51847PRTArtificial Sequencebinding peptide 184Thr Ser Leu
Val Pro Asp Lys1 51857PRTArtificial Sequencebinding peptide 185Thr
Thr Met Ala Tyr Val Ala1 51867PRTArtificial Sequencebinding peptide
186Thr Val Pro Met Arg Ser Leu1 518712PRTArtificial Sequencebinding
peptide 187Val Asp Arg Asn Gln Ser Leu Arg Ser Phe Xaa Thr1 5
101887PRTArtificial Sequencebinding peptide 188Val Gly Gln Gly Asn
Thr Ser1 51897PRTArtificial Sequencebinding peptide 189Val Leu
Pro Met Tyr Ser His1 51907PRTArtificial Sequencebinding peptide
190Trp Leu Arg Pro Xaa Leu His1 51917PRTArtificial Sequencebinding
peptide 191Trp Gln Leu Ala Arg Pro Lys1 51927PRTArtificial
Sequencebinding peptide 192Trp Gln Thr Xaa Leu Thr Asp1
519312PRTArtificial Sequencebinding peptide 193Trp Ser Asn Lys Pro
Leu Ser Pro Asn Asp Leu Arg1 5 101947PRTArtificial Sequencebinding
peptide 194Xaa Ala Leu Pro Trp Lys Ser1 51957PRTArtificial
Sequencebinding peptide 195Xaa Asp Ser Xaa Ser Xaa Xaa1
51967PRTArtificial Sequencebinding peptide 196Xaa Pro Thr Val Asp
Asn His1 51977PRTArtificial Sequencebinding peptide 197Xaa Pro Xaa
Xaa Val Phe Xaa1 51987PRTArtificial Sequencebinding peptide 198Tyr
Ala Asp Ser Val Gln Met1 51997PRTArtificial Sequencebinding peptide
199Tyr Pro Ala Pro Lys Pro Tyr1 520012PRTArtificial Sequencebinding
peptide 200Tyr Ser Ile Xaa Val Met Gly Tyr Tyr Thr Pro Tyr1 5
102017PRTArtificial Sequencebinding peptide 201Tyr Thr Lys Thr Ser
Gln Tyr1 52027PRTArtificial Sequencebinding peptide 202Ala Asp Lys
Thr Lys Asn Tyr1 52037PRTArtificial Sequencebinding peptide 203Gln
Tyr His Gly Pro Leu Pro1 52047PRTArtificial Sequencebinding peptide
204Thr Pro Pro Met Gly Arg His1 52057PRTArtificial Sequencebinding
peptide 205His Pro Thr Ala Gln Thr Leu1 52067PRTArtificial
Sequencebinding peptide 206Ser Asp Glu Ser Met Asn Met1
52077PRTArtificial Sequencebinding peptide 207Ser His Phe Ser Gly
Asn Arg1 52087PRTArtificial Sequencebinding peptide 208Asp His Asn
Gln Thr Asn Arg1 52097PRTArtificial Sequencebinding peptide 209Leu
His Thr His Ser Asn Thr1 52107PRTArtificial Sequencebinding peptide
210Asn Gly Asn Phe Asp Ser Ala1 52117PRTArtificial Sequencebinding
peptide 211Arg Pro Leu Met Ser Thr Gln1 52127PRTArtificial
Sequencebinding peptide 212Val Asn Asp Pro Thr Thr Ile1
52137PRTArtificial Sequencebinding peptide 213Thr Gly Asn Ser Ser
Gln Gln1 52147PRTArtificial Sequencebinding peptide 214Lys Ser Thr
Leu Tyr His Thr1 52157PRTArtificial Sequencebinding peptide 215Lys
Ala Ala His Asp Glu Gly1 52167PRTArtificial Sequencebinding peptide
216Trp His Thr Gly Pro Ser Glu1 52177PRTArtificial Sequencebinding
peptide 217Ile Thr Gln Glu Arg Asn Gln1 52187PRTArtificial
Sequencebinding peptide 218Gly Asn Asp Gln Val Ser Pro1
52197PRTArtificial Sequencebinding peptide 219Leu Thr Asp Ser Phe
Leu Gly1 52207PRTArtificial Sequencebinding peptide 220Pro Tyr Thr
Trp His Leu Glu1 52217PRTArtificial Sequencebinding peptide 221Ala
Thr Asp Asn Thr Leu Gln1 52227PRTArtificial Sequencebinding peptide
222Pro Val Ser Met Ile Ser Thr1 52237PRTArtificial Sequencebinding
peptide 223Leu Asn Lys Thr Ser Pro Asn1 522412PRTArtificial
Sequencebinding peptide 224Ser Ser Tyr Gln Ile Asn Thr Thr Pro Ala
Leu Pro1 5 1022512PRTArtificial Sequencebinding peptide 225Leu Pro
Leu Gln Pro Leu Met Pro Pro Leu Asn Gln1 5 1022612PRTArtificial
Sequencebinding peptide 226Phe Tyr Phe Pro Gln Asn Leu Val Tyr Gln
Ala Gly1 5 1022712PRTArtificial Sequencebinding peptide 227Asn Ser
Ser Pro Phe Ala Thr Met Pro Asn Ala Leu1 5 1022812PRTArtificial
Sequencebinding peptide 228Thr Cys Asn Ala Met Ser Ser Leu Cys Asp
Pro Pro1 5 1022912PRTArtificial Sequencebinding peptide 229Ser Pro
Leu Pro Pro Leu Val Gly Ser Leu Leu Lys1 5 1023012PRTArtificial
Sequencebinding peptide 230Phe Pro Thr Lys His Thr Leu Ser Thr Thr
Ile Tyr1 5 1023112PRTArtificial Sequencebinding peptide 231His Gly
Pro Arg Pro Pro Gly Met Thr Leu Pro Ile1 5 1023211PRTArtificial
Sequencebinding peptide 232Ser Pro Leu Leu Thr Tyr Lys Gln Gln Ala
Leu1 5 1023312PRTArtificial Sequencebinding peptide 233Lys Leu Pro
Tyr Pro Phe Pro Pro Glu Ala Met Val1 5 1023412PRTArtificial
Sequencebinding peptide 234His Pro Phe Leu Pro Pro Ser Lys Thr Ala
Pro Pro1 5 1023512PRTArtificial Sequencebinding peptide 235Trp Thr
Xaa Cys Val Glu Cys Thr Phe Ala Thr Pro1 5 1023612PRTArtificial
Sequencebinding peptide 236Gly Ala Lys His Tyr Ala Arg Val Ala Ala
Glu Phe1 5 1023712PRTArtificial Sequencebinding peptide 237Gly Ile
Met Gln Ser Thr Pro Pro Ala Asn Gln Gln1 5 1023812PRTArtificial
Sequencebinding peptide 238Tyr Gly Thr Gln Gln Gln Asp Arg Leu His
Lys Pro1 5 1023912PRTArtificial Sequencebinding peptide 239Val Asp
Glu Phe Leu His Ala Met Pro Leu Asn Ala1 5 1024012PRTArtificial
Sequencebinding peptide 240His Met Pro His Pro Ala Thr Val His Leu
Leu Trp1 5 1024112PRTArtificial Sequencebinding peptide 241Arg Ala
Ala Thr Ala Glu Leu Pro Gly Gly Arg Val1 5 1024212PRTArtificial
Sequencebinding peptide 242Leu Ile Glu Pro Tyr Thr Arg Ser Ala Asn
Ser Phe1 5 1024312PRTArtificial Sequencebinding peptide 243Asp Val
Asp Gln Leu Arg Ser Ala Val Trp Ser Arg1 5 102447PRTArtificial
Sequencebinding peptide 244Leu Ser Val Thr Thr Asn Thr1
52457PRTArtificial Sequencebinding peptide 245Asp Ser Leu Phe Lys
Trp Thr1 52467PRTArtificial Sequencebinding peptide 246Phe Glu Thr
Lys Ala Asn Asp1 52477PRTArtificial Sequencebinding peptide 247Asn
Val Asn Asn His Ile His1 52487PRTArtificial Sequencebinding peptide
248Gln Pro Ala Lys Gly Val Leu1 52497PRTArtificial Sequencebinding
peptide 249Asp Tyr Ala His Gly Asn Thr1 52507PRTArtificial
Sequencebinding peptide 250Ser Arg Ser Glu Leu Pro Leu1
52517PRTArtificial Sequencebinding peptide 251Ala Asp Arg Leu Arg
Pro Thr1 52527PRTArtificial Sequencebinding peptide 252His Ser Pro
Gln Met Gln Ser1 52537PRTArtificial Sequencebinding peptide 253Asn
Leu Ala Arg Asp Gly Thr1 52547PRTArtificial Sequencebinding peptide
254Thr Gly Asn Lys Ser Ser Met1 52557PRTArtificial Sequencebinding
peptide 255Thr Lys Asp Ala Trp Pro Ser1 52567PRTArtificial
Sequencebinding peptide 256Ser Pro Ala Leu Val Asn Ser1
52577PRTArtificial Sequencebinding peptide 257Val Asn Ser Asp Asn
Ala Tyr1 52587PRTArtificial Sequencebinding peptide 258Thr Ala Glu
Val Thr Arg Gly1 52597PRTArtificial Sequencebinding peptide 259Thr
Asn Lys Ile Pro Pro Leu1 52607PRTArtificial Sequencebinding peptide
260Thr Asn Pro Asn His Ile Met1 52617PRTArtificial Sequencebinding
peptide 261Ser Ser Ala Thr Ser Ile Thr1 52627PRTArtificial
Sequencebinding peptide 262Gln Pro Leu Lys Thr Lys Gln1
52637PRTArtificial Sequencebinding peptide 263Ile Glu Ser Arg Ser
Met Gln1 52647PRTArtificial Sequencebinding peptide 264Ser Phe Lys
Ser Met Thr Phe1 52657PRTArtificial Sequencebinding peptide 265His
Ser Leu Met Met Pro Asn1 52667PRTArtificial Sequencebinding peptide
266Thr Lys Ser Pro Thr Ala Ile1 52677PRTArtificial Sequencebinding
peptide 267Leu Ala Ser Glu Asn Met Asn1 52687PRTArtificial
Sequencebinding peptide 268Pro Pro His Ser His Gln Leu1
52697PRTArtificial Sequencebinding peptide 269Gln Val Asn Tyr Thr
Ser Val1 52707PRTArtificial Sequencebinding peptide 270Lys Ser Pro
Glu Tyr Pro Phe1 52717PRTArtificial Sequencebinding peptide 271Lys
Ala Pro His Gln Lys Ala1 52727PRTArtificial Sequencebinding peptide
272Thr Arg Ser Pro Ser Tyr Leu1 52737PRTArtificial Sequencebinding
peptide 273Pro Asn Pro Trp Asn Ala Phe1 52747PRTArtificial
Sequencebinding peptide 274Pro Ser Ser His Ser Tyr Arg1
52757PRTArtificial Sequencebinding peptide 275Lys Val Asn Met Leu
His Asp1 52767PRTArtificial Sequencebinding peptide 276Thr Ala His
Ala Met His Leu1 52777PRTArtificial Sequencebinding peptide 277His
Pro Gly Leu Ser Asn Lys1 52787PRTArtificial Sequencebinding peptide
278Val Pro Ile Asn Ser Ser Val1 52797PRTArtificial Sequencebinding
peptide 279Lys Ser Asn Asn Thr Gly Tyr1 52807PRTArtificial
Sequencebinding peptide 280Leu Trp Asn Ala Lys Leu Ala1
52817PRTArtificial Sequencebinding peptide 281Gln Met Thr Gln Thr
Gln Ser1 52827PRTArtificial Sequencebinding peptide 282Thr Ser Gly
Pro His Pro Leu1 52837PRTArtificial Sequencebinding peptide 283Asn
Glu Ala Leu Gly His Leu1 52847PRTArtificial Sequencebinding peptide
284Ser Gly Leu Ser Lys Leu Asn1 52857PRTArtificial Sequencebinding
peptide 285Lys His Ala Asp Ser Thr Ser1 52867PRTArtificial
Sequencebinding peptide 286Ser Thr Ser Gln His Asn Val1
52877PRTArtificial Sequencebinding peptide 287Thr Thr Gln Thr Asn
Lys Asp1 52887PRTArtificial Sequencebinding peptide 288Asn Thr Ala
Ala Thr Gly Thr1 52897PRTArtificial Sequencebinding peptide 289Pro
Ala Thr Asn Pro Asn His1 52907PRTArtificial Sequencebinding peptide
290Leu Ala Glu Thr His Ser Ser1 52917PRTArtificial Sequencebinding
peptide 291His Thr Asp Thr Ser Pro Gln1 52927PRTArtificial
Sequencebinding peptide 292Ser Pro Leu Tyr His Asp Arg1
52937PRTArtificial Sequencebinding peptide 293Leu Lys Tyr Leu Glu
Arg Asp1 52947PRTArtificial Sequencebinding peptide 294Leu Ser Glu
Ala Pro Gly Ile1 52957PRTArtificial Sequencebinding peptide 295Glu
Arg Gln Asn Asn Met Asn1 52967PRTArtificial Sequencebinding peptide
296Asn Ala Phe Glu Ser Leu Phe1 52977PRTArtificial Sequencebinding
peptide 297Cys Tyr Ile Pro Thr Pro Arg1 52987PRTArtificial
Sequencebinding peptide 298Asn Ser Tyr Asn Ser Gly Leu1
52997PRTArtificial Sequencebinding peptide 299Asp Pro Gln Ala Asn
Leu Thr1 53007PRTArtificial Sequencebinding peptide 300Arg Gln Ala
Asn Leu Thr Gln1 53017PRTArtificial Sequencebinding peptide 301Leu
Asp Gln His Ser Met Lys1 53027PRTArtificial Sequencebinding peptide
302His Asn Met His Gln Ala Val1 53037PRTArtificial Sequencebinding
peptide 303Leu Asn Thr Leu Leu Gly Thr1 53047PRTArtificial
Sequencebinding peptide 304Leu Leu Pro Arg Leu His Asp1
53057PRTArtificial Sequencebinding peptide 305Pro His His Lys Met
Gln Asn1 53067PRTArtificial Sequencebinding peptide 306Pro Gly Glu
Ala Arg Gly Glu1 53077PRTArtificial Sequencebinding peptide 307Gly
Ser His Ser Pro Pro Gln1 53087PRTArtificial Sequencebinding peptide
308Lys Leu Gln Ala His Pro Asn1 53097PRTArtificial Sequencebinding
peptide 309Phe Thr Met Asn Asp Ile Arg1 53107PRTArtificial
Sequencebinding peptide 310Pro Ser Thr Thr Lys His Gly1
53117PRTArtificial Sequencebinding peptide 311Asn Ser Thr Arg Thr
Phe Ala1 53127PRTArtificial Sequencebinding peptide 312Pro Ser His
Thr Asn Val Asn1 53137PRTArtificial Sequencebinding peptide 313Lys
Pro Thr Phe Ile Arg Ala1 53147PRTArtificial Sequencebinding peptide
314Asp Pro Arg Lys Ser Ala Gln1 53157PRTArtificial Sequencebinding
peptide 315Gly Leu Thr Arg His Gln Ala1 53167PRTArtificial
Sequencebinding peptide 316Ser Ala Ser Thr Pro Arg Ala1
53174PRTArtificial Sequencerepeatable motif 317Ile Asn Ala
Gln13184PRTArtificial Sequencerepeatable motif 318Lys Thr Pro
Ser13194PRTArtificial Sequencerepeatable motif 319Asn Ser Ser
Ser13204PRTArtificial Sequencerepeatable motif 320Asn Thr Ser
Pro13214PRTArtificial Sequencerepeatable motif 321Ser Asn Ala
Thr13225PRTArtificial Sequencerepeatable motif 322Gln Ala Asn Leu
Thr1 53235PRTArtificial Sequencerepeatable motif 323Thr Asn Pro Asn
His1 53244PRTArtificial Sequencerepeatable motif 324Ala Asn Leu
Thr13254PRTArtificial Sequencerepeatable motif 325Asn Pro Asn
His13264PRTArtificial Sequencerepeatable motif 326Gln Ala Asn
Leu13274PRTArtificial Sequencerepeatable motif 327Thr Asn Pro
Asn13284PRTArtificial Sequencerepeatable motif 328Cys Ser Pro
Met13294PRTArtificial Sequencerepeatable motif 329Cys Ser Pro
Leu13305PRTArtificial Sequencerepeatable motif 330Cys Lys Thr Pro
Ser1 53314PRTArtificial Sequencerepeatable motif 331Cys Thr Thr
Thr13324PRTArtificial Sequencerepeatable motif 332Thr Thr Ala
Cys1
* * * * *