U.S. patent application number 10/533701 was filed with the patent office on 2006-08-03 for skin or hair binding peptides.
Invention is credited to Giselle Janssen, Christopher Murray, Deborah Winetzky.
Application Number | 20060171885 10/533701 |
Document ID | / |
Family ID | 32393497 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060171885 |
Kind Code |
A1 |
Janssen; Giselle ; et
al. |
August 3, 2006 |
Skin or hair binding peptides
Abstract
The invention is directed to peptides. Specifically, the
invention is directed to peptides which bind skin and do not bind
hair. Alternatively, the invention is drawn to peptides which bind
hair and do not bind skin.
Inventors: |
Janssen; Giselle; (San
Diego, CA) ; Murray; Christopher; (Soquel, CA)
; Winetzky; Deborah; (Foster City, CA) |
Correspondence
Address: |
Kamrin T MacKnight;Genencor International
925 Page Mill Road
Palo Alto
CA
94304-1013
US
|
Family ID: |
32393497 |
Appl. No.: |
10/533701 |
Filed: |
November 12, 2003 |
PCT Filed: |
November 12, 2003 |
PCT NO: |
PCT/US03/36234 |
371 Date: |
January 12, 2006 |
Current U.S.
Class: |
424/1.69 |
Current CPC
Class: |
A61Q 5/02 20130101; C07K
7/06 20130101; A61K 8/64 20130101; C12N 15/1037 20130101; C07K
1/047 20130101; C07K 7/08 20130101 |
Class at
Publication: |
424/001.69 |
International
Class: |
A61K 51/00 20060101
A61K051/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 25, 2002 |
US |
60429051 |
Claims
1. A skin binding peptide including (a) any one of SEQ ID NOs. 1-24
or (b) an amino acid sequence having at least 50% sequence identity
to any one sequence of SEQ ID NOs. 1-24 and including a sequence
cluster selected from the group consisting of APQQRPMXTXXX (SEQ ID
NO. 25); PPWXXXL (SEQ ID NO. 26); XXTXLTS (SEQ ID NO. 27); XPPLLXL
(SEQ ID NO. 28); SXPSGAX (SEQ ID NO. 29); XQATFXXNXXXX (SEQ ID NO.
30); VXTSQLXXXXXX (SEQ ID NO. 31); LXXXRMK (SEQ ID NO. 32), and
HXXXYLT (SEQ ID NO. 33), wherein X represents any L-amino acid.
2. The skin binding peptide according to claim 1, wherein the
peptide has at least 90% sequence identity to any of SEQ ID NOs.
1-24.
3. The skin binding peptide according to claim 1, wherein the
peptide has at least 95% sequence identity to any of SEQ ID NOs.
1-24.
4. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid SEQ ID NO1.
5. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid SEQ ID NO. 3.
6. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid SEQ ID NO. 5.
7. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid SEQ ID NO. 6.
8. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid SEQ ID NO. 8.
9. The skin binding peptide according to claim 1, wherein the
peptide includes amino acid sequence SEQ ID NO. 15.
10. The skin binding peptide according to claim 1, wherein the
peptide includes the sequence cluster APQQRPMXTXXX (SEQ ID NO.
25).
11. The skin binding peptide according to claim 1, wherein the
peptide includes the sequence cluster XQATFXXNXXXX (SEQ ID NO.
30).
12. The skin binding peptide according to claim 1, wherein the
peptide includes the sequence cluster LXXXRMK (SEQ ID NO. 32).
13. The skin binding peptide according to claim 1, wherein the
peptide includes a C-C derivative.
14. A composition comprising any one of the skin binding peptides
according to claim 1.
15. A hair binding peptide including (a) any one of SEQ ID NOs.
34-56 or (b) an amino acid sequence having at least 50% sequence
identity to any one sequence of SEQ ID NOs. 34-56 and including a
sequence cluster selected from the group consisting of NTPXXNX (SEQ
ID NO. 57); PXXXLST (SEQ ID NO. 58); TXPTHR (SEQ ID NO. 59);
LXTXSTP (SEQ ID NO. 60); and TPLTXXT (SEQ ID NO. 61) and XQXHNPP
(SEQ ID NO. 62), wherein X represents any L-amino acid.
16. The hair binding peptide according to claim 1, wherein said
peptide has at least 90% sequence identity to any of SEQ ID NOs.
34-56.
17. The hair binding peptide according to claim 1, wherein the
peptide has at least 95% sequence identity to any of SEQ ID NOs.
34-56.
18. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster NTPXXNX (SEQ ID
NO. 57).
19. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster PXXXLST (SEQ ID
NO. 58).
20. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster TXPTHR (SEQ ID
NO. 59).
21. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster LXTXSTP (SEQ ID
NO. 60).
22. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster TPLTXXT (SEQ ID
NO. 61).
23. The hair binding peptide according to claim 15, wherein the
hair binding peptide includes the sequence cluster XQXHNPP (SEQ ID
NO. 62).
24. A composition comprising at least one hair binding peptide
according to claim 15.
Description
BACKGROUND OF THE INVENTION
[0001] Various methods may be used for the selection and
identification of compounds capable of binding specifically to a
target in the presence of undesired background targets
(anti-targets) using libraries of similar compounds. This invention
is directed to peptides that bind specifically to a target such as
skin or hair in the presence of an anti-target. The anti-target is
hair when the target is skin and the anti-target is skin when the
target is hair.
[0002] The literature is replete with examples of recent advances
in methods for screening large library pools of compounds,
especially peptides. Methods for screening these compounds to
identify molecules that bind to a preselected target have also been
advanced. One well-known method is biopanning which was originally
developed by Smith, G. P., (1985), Science 228:1315. Biopanning in
its simplest form is an in vitro selection process in which a
library of phage-displayed peptides is incubated with a target. The
target and phage are allowed to bind and unbound phage are washed
away. The specifically bound phage are then acid eluted. The eluted
pool of phage is amplified in vivo and the process is repeated.
After a number of rounds individual clones are isolated and
sequenced.
[0003] A number of variations of the biopanning technique first
introduced by Smith have been described, and reference is made to
Christian et al., (1992) J. Mol. Bio., 227:711; Cwirla et al.,
(1990) Proc. Natl. Acad. Sci. USA, 87:6378; Cull et al., (1992)
Proc. Natl. Acad. Sci. USA, 89:1865; Huls et al., (1996) Nature
Biotechnol., 7:276; and Bartoli et al., (1998) Nature Biotechnol.,
16:1068.
[0004] Huls et al., 1996 supra, describe a method comprising flow
cytometry-based subtractive selection of phage antibody on intact
tumor cells. The phage-displayed antibodies remain bound to the
target during the flow-cytometric selection. However, prior to
amplification the cell-bound phages are eluted from the target. WO
98/54312 discloses selection of antibodies under mild conditions
with high affinities for antigens using antibody libraries
displayed on ribosomes.
[0005] Balass et al., (1996) Anal. Biochem., 243:264, describe the
selection of high-affinity phage-peptides from phage-peptide
libraries using a biotinylated target immobilized on a
nitrostreptavidin matrix. Other targeting methods include, for
example, SELEX (U.S. Pat. No. 5,475,096) and selective targeting
which includes deselection as disclosed in WO 01/79479.
SUMMARY OF THE INVENTION
[0006] In a first aspect the invention concerns a peptide which
binds to skin but not to hair, and in a second aspect the invention
concerns a peptide which binds to hair but not to skin.
[0007] In a third aspect the invention concerns a skin binding
peptide including (a) any one of SEQ ID NOs. 1-24 or (b) an amino
acid sequence having at least 50% sequence identity to any one
sequence of SEQ ID NOs. 1-24 and including a sequence cluster
selected from the group consisting of APQQRPMXTXXX (SEQ ID NO. 25);
PPWXXXL (SEQ ID NO. 26); XXTXLTS (SEQ ID NO. 27); XPPLLXL (SEQ ID
NO. 28); SXPSGAX (SEQ ID NO. 29); XQATFXXNXXXX (SEQ ID NO. 30);
VXTSQLXXXXXX (SEQ ID NO. 31); LXXXRMK (SEQ ID NO. 32), and HXXXYLT
(SEQ ID NO. 33), wherein X represents any L-amino acid.
[0008] In one embodiment a skin binding peptide of the invention
includes a C-C derivative. Particularly preferred skin binding
peptides of the invention include a peptide having the amino acid
sequence of SEQ ID NO. 1; a peptide including the sequence cluster
of sequence XQATFXXNXXXX (SEQ ID NO. 30); a peptide having the
amino acid sequence of SEQ ID NO. 5; a peptide having the sequence
cluster of sequence LXXXRMK (SEQ ID NO. 32) and a peptide having
the sequence cluster of APQQRPMXTXXX (SEQ ID NO. 25). In another
embodiment the invention concerns a composition comprising one or
more skin binding peptides as disclosed herein.
[0009] In a fourth aspect the invention concerns a hair binding
peptide including (a) any one of SEQ ID NOs. 34-56 or (b) an amino
acid sequence having at least 50% sequence identity to any one
sequence of SEQ ID NOs. 34-56 and including a sequence cluster
selected from the group consisting of NTPXXNX (SEQ ID NO. 57);
PXXXLST (SEQ ID NO. 58); TXPTHR (SEQ ID NO. 59); LXTXSTP (SEQ ID
NO. 60); and TPLTXXT (SEQ ID NO. 61) and XQXHNPP (SEQ ID NO. 62),
wherein X represents any L-amino acid.
[0010] In one embodiment a hair binding peptide of the invention
includes a C-C derivative. In another embodiment the invention
concerns a composition comprising one or more hair binding peptides
as disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a general schematic diagram of a targeting method
which maybe used to select for the skin or hair binding peptides
identified herein. The method comprises the steps of, a) selection
against anti-targets which provides a library of ligands depleted
of anti-target bound ligands, b) selection for the target by
formation of a target-bound ligand complex, c) separation of the
target-bound ligand complex, d) identification of the target-bound
ligands, and e) optionally sequencing the target-bound ligands,
exposing the target-bound ligands to additional rounds of selective
targeting, and/or diversification.
[0012] FIG. 2 illustrates the stability of phage-peptide libraries
(Ph.D. 7, Ph.D. C7C and Ph.D.12) in Neutrogena.RTM. Shower
Shampoo.
[0013] FIG. 3 illustrates the stability of phage-peptide libraries
(Ph.D. 7, Ph.D. C7C and Ph.D.12) in Neutrogena.RTM. Bath Gel.
DETAILED DESCRIPTION OF THE INVENTION
[0014] 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
purposes of the present invention, the following terms are used to
describe the invention herein.
[0015] The term "ligand" refers to a molecule or compound that is
recognized by a particular target or anti-target. The term is
independent of molecular size or compositional feature. The ligand
may serve as a substrate for an enzyme-catalyzed reaction, as an
agonist, as an antagonist, act as a signal messenger, or stimulate
or inhibit metabolic pathways. Ligands may be nucleic acids,
peptides, peptide derivatives, peptidomimetics, polypeptides, small
organic molecules, carbohydrates and other molecules that are
isolated from a candidate mixture that acts on a target in a
desirable manner. Preferably the desirable manner is binding the
target.
[0016] The term "library" refers to a collection of chemical or
biological entities that can be created in a single reservoir and
simultaneously screened for a desired property. As used herein a
library can have a minimum size of at least two members and may
contain as many as 10.sup.15 members. In one aspect, the library
has at least 10.sup.2 members. In another aspect, the library has
at least 10.sup.3 members. In yet another aspect, the library has
at least 10.sup.6 members. In a further aspect, the library has at
least 10.sup.9 members. The size of a library refers to the total
number of entities comprising the library whether the members are
the same or different.
[0017] A "peptide library" refers to a set of peptides and to the
peptides and any fusion proteins containing those peptides.
Stochastic or random processes may be used to construct random
peptides. The term "random" does not mean that the library
composition is not known.
[0018] The term "peptide" refers to an oligomer in which the
monomeric 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 identified according to the
invention are preferably less than 50 amino acids in length, more
preferably less than 30 amino acids in length, also preferably less
than 25 amino acids in length, and preferably less than 20 amino
acids in length. In one embodiment the identified binding peptides
are between 4 and 20 and also between 6 and 15 amino acids in
length. However, in general peptides may be up to 100 amino acids
in length. Peptides that are longer than 100 amino acids in length
are generally referred to as polypeptides. Standard abbreviations
for amino acids are used herein. (See Singleton et al., (1987)
Dictionary of Microbiology and Molecular Biology, Second Ed., page
35, incorporated herein by reference). The term "protein" is well
known and refers to a large polypeptide.
[0019] A "binding peptide" according to the invention is a peptide
that binds to a target (skin or hair) with a binding affinity of at
least about 10.sup.-2 M, at least about 10.sup.-3 M, at least about
10.sup.-4 M, at least about 10.sup.-5 M, at least about 10.sup.-7
M, at least about 10.sup.-9 M, and preferably between about
10.sup.-2 M to 10.sup.-15 M, between about 10.sup.-2 M to
10.sup.-10M and between 10.sup.-3 to 10.sup.-9M.
[0020] The term "nucleic acid" means DNA, RNA, single-stranded or
double-stranded and chemical modifications thereof. Modifications
may include but are not limited to modified bases, backbone
modifications, methylations, unusual base pairing modifications,
and capping modifications.
[0021] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
peptide encoding nucleotide sequences may be produced. "Percent
sequence identity" with respect to a peptide or nucleic acid
sequence refers to the percent of residues or codons that are
identical in two sequences. Peptide or polynucleotides according to
the invention may have at least 50%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%
and at least 95% sequence identity to a reference sequence when
optimally aligned. Optimal alignment of the sequences may be
conducted by various known methods and computerized implementation
of known algorithms (e.g. BLAST, TFASTA, BESTFIT, such as in the
Wisconsin Genetics Software Package, Release 7.0, Genetics Computer
Group, Madison, Wis.).
[0022] The term "target" or "anti-target" refers to molecules or
heterogeneous molecules that have a binding affinity as defined
herein, for a given ligand. Both target and anti-targets may be
naturally occurring or synthetic molecules or heterogeneous
molecules. In a preferred embodiment the target is skin or hair.
Further when the target is skin, the anti-target is hair and when
the target is hair, the anti-target is skin.
[0023] The binding affinity of a ligand for its target or
anti-target may be described by the dissociation constant
(K.sub.D), concentration needed for 50% effective binding
(EC.sub.50), or concentration needed for 50% inhibition of binding
of another compound that binds to the target (IC.sub.50). K.sub.D
is defined by k.sub.off/k.sub.on. The k.sub.off value defines the
rate at which the target-ligand complex breaks apart or separates.
This term is sometimes referred to in the art as the kinetic
stability of the target-ligand complex or the ratio of any other
measurable quantity that reflects the ratio of binding affinities,
such as an enzyme-linked immunosorbent assay (ELISA) signal or
radio-active label signal.
[0024] "Selectivity" is defined by the ratio of binding affinities
or k.sub.off for dissociation of the ligand-complex (target
K.sub.D/anti-target K.sub.D). The k.sub.on value describes the rate
at which the target and ligand combine to form the target-ligand
complex.
[0025] The term "contacting" is broadly defined to mean placing a
library of ligands and a target or anti-target in immediate
proximity or association and includes in vitro and in vivo contact.
The term includes touching, associating, joining, combining,
intravenous injection, oral administration, intraperitoneally,
topical application, intramuscular, inhalation, subcutaneous
application and the like.
[0026] The term "separating" as used herein means to select,
segregate, partition, isolate, collect, keep apart and disunite.
Separation methods are well known to those in the art. These
methods include affinity chromatography, washing, liquid transfer,
centrifugation, high-performance liquid chromatography (HPLC),
filtration, such as gel filtration, enzyme-linked immunosorbent
assays (ELISA), and fluorescence-activator cell sorting (FACS). The
choice of a separation method is well within the skill of one in
the art, and a variety of instruments used for these separation
methods are commercially available. (See Kenny and Fowell (eds)
(1992) Practical Protein Chromatography Methods in Molecular
Biology, vol. 11, Humana Press, Totowa N.J.).
[0027] "Amplifying" means a process or combination of process steps
that increases the amount or number of copies of a molecule or
class of molecules. In one aspect, amplification refers to the
production of additional copies of nucleic acid sequences that is
carried out using polymerase chain reaction (PCR) technology well
known in the art.
[0028] As used in the specification, the singular "a", "an" and
"the" include the plural references unless the context clearly
dictates otherwise. For example, the term "a ligand" may include a
plurality of ligands.
[0029] The following references describe the general techniques
employed herein: Sambrook et al., (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Innis et al., PCR Protocols--A Guide to Methods and
Applications (1990), Academic Press, Inc.; Kay et al., (1996) Phage
Display of Peptides and Proteins, Academic Press; Ausubel et al.,
(1987) Current Protocols in Molecular Biology, Greene-Publishing
& Wiley Interscience NY (Supplemented through 1999); Berger and
Kimmel, (1987) Methods in Enzymology, Vol. 152. Academic Press
Inc., San Diego, Calif.
[0030] The skin or hair binding peptides of the invention may be
obtained and identified using methods well known in the art. These
methods may include use of random peptide libraries, synthetic
peptide libraries, peptide loop libraries, antibody libraries and
protein libraries. These libraries as well as methods for making
the libraries are well known. Reference is made to Barbas, C. F.
(1993) Current Opinion in Biotech., 4:526; Cwirla et al., (1990)
supra; Scott and Smith, (1990) Science, 249:386; Cull et al.,
(1992) supra; Pinilla et al., (1994) Biochem. J. 301:847; Sambrook
et al., (1989) supra; Ausubel et al., (1987) supra; and Gubler and
Hoffman, (1983) Gene 25:263; U.S. Pat. No. 5,283,173; U.S. Pat. No.
5,270,181; U.S. Pat. No. 5,292,646; U.S. Pat. No. 5,605,793; U.S.
Pat. No. 5,733,731; Stemmer (1994), Proc. Natl. Aca. Sci. USA
91:10747; WO 97/22617; Foder et al., (1991) Science 251:767; Lam et
al., (1993) Bioorg. Med. Chem. Lett., 3:419; Tjoeng et al., (1990)
Int. J. Pept Protein Res. 35:141; and WO96/33010.
[0031] Various companies now make instrumentation to generate
combinatorial libraries from both solution and solid phase
synthesis (CombiChem Inc. (San Diego, Calif.); Advanced ChemTech
(Louisville); Zymark Corp. (MA); and Hewlett Packard (CA)).
[0032] Not only are standard methods for generating libraries of
ligands well known, but also ligand libraries may be obtained
commercially, for example from Sigma (St. Louis Mo.) or from
various public sources such as American Type Culture Collection
(ATCC) and the National Institute of Health (NIH).
[0033] Screening techniques may include yeast display, ribosome
display, biopanning and acid elution. One general method for
screening a library of ligands having a binding affinity and
selectivity for a selected target includes preparing or obtaining a
library of ligands, preferably peptides of different sequences and
more preferably a random peptide library. Deselecting ligands that
bind with an anti-target by contacting the ligand library with an
anti-target under conditions favorable for binding between the
ligands of the library and the anti-target; allowing the
anti-target to bind with the ligands; and separating the
anti-target non-binders (unbound ligands) from the anti-target
ligand bound molecules and any free ligands. Contacting the
anti-target non-binders with a selected target under suitable
conditions and allowing them to bind. Ligands with an affinity for
the target will bind to form a target-bound ligand complex. The
removal of ligands bound to the anti-target and removal of weak
target-bound ligands may generally be referred to as library
depletion. The target-bound ligand complex is then separated from
the remaining mixture including the unbound ligands. The
target-bound ligand complex or the target-bound ligands may
optionally be subjected to further rounds of selection (FIG.
1).
[0034] Once selected, a binding peptide may be sequenced, amplified
or produced in bulk by any one of a number of standard techniques.
Some of these techniques include polymerase chain reaction (PCR),
direct amino acid sequencing of the selected peptide by using
peptide sequencers, mass spectrophotometry (MS), surface plasmon
resonance, immunoprecipitation and nuclear magnetic resonance (NMR)
spectroscopy. Reference is made to U.S. Pat. No. 4,683,202; Szabo
et al., (1995) Curr. Opin. Struct. Bio. 5:699; Harlow et al.,
(1999) Using Antibodies, A Laboratory Manual, Cold Spring Harbor
Press; Hajduk et al., (1999) J. Med Chem., 42:2315; Cao et al.,
(1997) Techniques in Protein Chemistry VIII, Academic Press pages
177-184; Youngquist et al., (1995) J. Am. Chem. Soc. 117:3900;
Cheng et al., (1995) J. Am. Chem. Soc., 117:8859; Walk et al.,
(1999) Angew. Che. Int. Ed., 38:1763; Wu et al., (1997) in
Chemistry and Biology, vol. 14(9):653; Marshall et al., (1998),
Mass Spectrometry Reviews 17:1; and Nelson et al., (1999) J. Mol.
Recognition, 12:77.
[0035] The binding peptides may be produced recombinantly using
genetic engineering or the peptide may be chemically
synthesized.
[0036] In one embodiment, the peptide binding affinity for the
target according to the present invention for K.sub.D, EC.sub.50 or
IC.sub.50 is in the range of between about 10.sup.-7 M to
10.sup.-15 M, although higher or low binding affinities may be
achieved. In one aspect, the binding affinity is in the range of at
least about 10.sup.-2 M, at least about 10.sup.-3 M, at least about
10.sup.-4 M, at least about 10.sup.-5 M, at least about 10.sup.-7
M, at least about 10.sup.-9 M and also at least about 10.sup.-12 M.
In another embodiment, the affinity is less than about 10.sup.-7 M.
In another aspect, k.sub.off values for the ligand-target complex
will be less than about 10.sup.-2 sec.sup.-1, less than 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. The binding peptides of
the invention will not bind with any significance to the
anti-target. While not meant to limit the invention, a preferred
binding ligand may have a K.sub.D for the anti-target greater than
about 10.sup.-4 M, and preferably greater than about 10.sup.-1
M.
[0037] The binding peptides according to the invention may be
characterized not only by the binding affinity of the ligand to a
target, but also may be characterized by the selectivity of the
ligand-target complex. The selectivity of ligand binding for a
target compared to ligand binding to an anti-target can be defined
by a ratio of K.sub.D, EC.sub.50 or IC.sub.50 in the range of about
1.5:1 to 500:1. In one aspect, selectivity is at least about 2:1,
at least about 3:1, at least about 5:1, at least about 10:1, at
least about 20:1, at least about 30:1, at least about 50:1, and at
least about 100:1.
[0038] Methods for measuring binding affinities and selectivity are
well known in the art, and these methods include but are not
limited to measurement by radio-labeled release and competition
assay; by isothermal titration calorimetry; biosensor binding
assays (Morton & Myszka, (1998) Methods Enzymol. 295:268-294);
by fluorescence and chemiluminescence spectroscopy; and by mass
spectrophotometry (Gao et al., (1996), J. Med, Chem., 39:1949).
[0039] In one embodiment, preferred skin binding peptides according
to the invention are listed in Table 1. TABLE-US-00001 TABLE 1 Skin
binding peptides KQATFPPNPTAY SEQ ID NO. 1 QATFMYN SEQ ID NO. 2
HGHMVSTSQLSI SEQ ID NO. 3 VLTSQLPNHSM SEQ ID NO. 4 LSPSRMK SEQ ID
NO. 5 LPIPRMK SEQ ID NO. 6 HSTAYLT SEQ ID NO. 7 HQRPYLT SEQ ID NO.
8 APQQRPMKTFNT SEQ ID NO. 9 APQQRPMKTVQY SEQ ID NO. 10 PPWLDLL SEQ
ID NO. 11 PPWTFPL SEQ ID NO. 12 SVTHLTS SEQ ID NO. 13 VITRLTS SEQ
ID NO. 14 FPPLLRL SEQ ID NO. 15 DLKPPLLALSKV SEQ ID NO. 16
SHPSGALQEGTF SEQ ID NO. 17 FPLTSKPSGACT SEQ ID NO. 18 DLKPPLLALSKV
SEQ ID NO. 19 PLLALHS SEQ ID NO. 20 YPISTQI SEQ ID NO. 21
YAKQHYPISTFK SEQ ID NO. 22 HSTAYLT SEQ ID NO. 23 STAYLVAMSAAP SEQ
ID NO. 24
[0040] Particularly preferred embodiments include skin binding
peptides having an amino acid sequence of SEQ ID NO. 1, SEQ ID NO.
3, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 15, and
particularly SEQ ID NO. 1 and SEQ ID NO. 5.
[0041] In another embodiment, skin binding peptides of the
invention have repeatable motifs. A repeatable motif is defined as
including at least three consecutive amino acid residues in a
peptide string and may include four, five, six, seven, eight or
nine consecutive amino acids residues wherein the repeatable motif
is found in at least two of the peptides listed in Table 1 or at
least two of the peptides listed in Table 2. Preferred repeatable
motifs for skin binding peptides include QATF, TSQL, RMK, YLT,
APQQRPM, PMKT, PPW, LTS, PPLL, APQQRMKT, PSGA, PLLAL, STAYL, and
YPIST.
[0042] In yet a further embodiment, skin binding and hair binding
peptides of the invention include sequence clusters. A sequence
cluster includes a repeatable motif as defined herein and 1 or 2
amino acid residues identical to amino acid residues found in the
binding peptides listed in Table 1 or Table 2 when the same
repeatable motif in each peptide is aligned and further including 1
to 8, preferably 1 to 3 intervening amino acid residues located
either before or after the repeatable motif.
[0043] Preferred sequence clusters for skin binding peptides
include the following: TABLE-US-00002 APQQRPMXTXXX SEQ ID NO. 25
PPWXXXL SEQ ID NO. 26 XXTXLTS SEQ ID NO. 27 XPPLLXL SEQ ID NO. 28
SXPSGAX SEQ ID NO. 29 XQATFXXNXXXX SEQ ID NO. 30 VXTSQLXXXXXX SEQ
ID NO. 31 LXXXRMK SEQ ID NO. 32 HXXXYLT SEQ ID NO. 33
X represents any L-amino acid. Particularly preferred skin binding
peptides are peptides having the sequence cluster of APQQRPMXTXXX
(SEQ ID NO. 25), the sequence cluster of XQATFXXNXXXX (SEQ ID NO.
30) or LXXXRMK (SEQ ID NO. 32).
[0044] In a further embodiment, preferred hair binding peptides
according to the invention are listed in Table 2. TABLE-US-00003
TABLE 2 Hair binding peptides NTPKENW SEQ ID NO. 34 NTPASNR SEQ ID
NO. 35 PRGMLST SEQ ID NO. 36 PPTYLST SEQ ID NO. 37 TIPTHRQHDYRS SEQ
ID NO. 38 TPPTHRL SEQ ID NO. 39 LPTMSTP SEQ ID NO. 40 LGTNSTP SEQ
ID NO. 41 TPLTGSTNLLSS SEQ ID NO. 42 TPLTKET SEQ ID NO. 43 KQSHNPP
SEQ ID NO. 44 QQSHNPP SEQ ID NO. 45 TQPHNPP SEQ ID NO. 46
STNLLRTSTVHP SEQ ID NO. 47 HTQPSYSSTNLF SEQ ID NO. 48 SLLSSHA SEQ
ID NO. 49 QQSSISLSSHAV SEQ ID NO. 50 NASPSSL SEQ ID NO. 51 HSPSSLR
SEQ ID NO. 52 K H/R/N SHHTH SEQ ID NO. 53 E H/R/N SHHTH SEQ ID NO.
54 SHHTHYGQPGPV SEQ ID NO. 55 LESTSLL SEQ ID NO. 56
[0045] The binding peptides identified as SEQ ID NO: 53 and SEQ ID
NO. 54 may include histidine (H), arginine (R) or asparagine (N) as
the second amino acid in the peptide string.
[0046] In another embodiment, hair binding peptides of Table 2 have
repeatable motifs. Preferred repeatable motifs for hair binding
peptides include STNL, LSSHA, SPSSL, SHHTH, NTP, LST, PTHR, STP,
TPLT and HNPP.
[0047] In yet a further embodiment hair binding peptides of the
invention include sequence clusters. Preferred sequence clusters
for hair binding peptides include the following: TABLE-US-00004
NTPXXNX SEQ ID NO. 57 PXXXLST SEQ ID NO. 58 TXPTHRX SEQ ID NO. 59
LXTXSTP SEQ ID NO. 60 TPLTXXT SEQ ID NO. 61 XQXHNPP SEQ ID NO.
62
X represents any L amino acid.
[0048] In a further embodiment a binding peptide according to the
invention includes peptides having a sequence cluster or repeatable
motif as disclosed above, wherein the peptide has at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, and at least 85% amino acid sequence identity with a
reference binding peptide of Table 1 or Table 2. The peptide, which
includes a sequence cluster or repeatable motif, will also have a
binding affinity for the same target as the reference peptide in
the range of 10.sup.-2 to 10.sup.-15M, at least about 10.sup.-2M,
at least about 10.sup.-3M, at least about 10.sup.-5M, at least
about 10.sup.-7M, and at least about 10.sup.-9M. Preferably the
binding affinity will be essentially the same or greater than the
binding affinity of the reference binding peptide.
[0049] Additionally a skin or hair binding peptide according to the
invention may include a cysteine (C) residue on either or both ends
of the peptide. These peptides are more specifically referred to
herein as C-C derivatives. Nonlimiting examples of C-C derivative
skin binding peptides include C-SEQ ID NO. 2-C; C-SEQ ID NO. 5-C;
C-SEQ ID NO. 6-C; C-SEQ ID NO. 7-C; C-SEQ ID NO.8-C; C-SEQ ID NO.
11-C; C-SEQ ID NO. 12-C; C-SEQ ID NO. 13-C; C-SEQ ID NO. 14-C;
C-SEQ ID NO. 15-C; C-SEQ ID NO. 20-C; C-SEQ ID NO. 21-C; and C-SEQ
ID NO. 23-C. Nonlimiting examples of C-C derivative hair binding
peptides include C-SEQ ID NO. 34-C; C-SEQ ID NO. 35-C; C-SEQ ID NO.
36-C; C-SEQ ID NO. 37-C; C-SEQ ID NO. 39-C; C-SEQ ID NO. 40-C;
C-SEQ ID NO. 41-C; C-SEQ ID NO. 43-C; C-SEQ ID NO. 46-C; C-SEQ ID
NO. 49-C; C-SEQ ID NO. 51-C; C-SEQ ID NO. 52-C; and C-SEQ ID NO.
56-C. A binding peptide which comprises a sequence cluster may also
include a cysteine residue on either or both ends of the
peptide.
[0050] A linker molecule (also sometimes referred to in the art as
a spacer moiety) may be added to either end of a binding peptide
according to the invention. A linker molecule may be any carbon
containing molecule such as, a short peptide, for example GGH,
GGGK, and GGHGG; a carbon chain, for example (CH.sub.2)n wherein n
equals 1 to 10; a polymer, for example PEG(CH.sub.2--O)n wherein n
equals 2-20; a sugar; a lipid or the like.
[0051] In one application, skin or hair binding peptides of the
invention may be used in is compositions for personal care
applications. These compositions may take the form of lotions,
creams, gels, sprays, shampoos and conditioners and the like.
[0052] Non-limiting examples of personal care applications which
include a binding peptide of the invention are the following: a)
using a skin binding peptide with an emollient which may result in
the enhancement of the moisturizing properties of the emollient; b)
combining a skin binding peptide with a bleaching or tanning agent
which may result in the enhancement of skin bleaching or tanning
properties; c) combining a skin binding peptides with a sunscreen
for topical application; and d) combining a hair binding peptide
with a dye or oxidizing agent wherein the hair coloring properties
of the hair coloring formulation may be enhanced.
[0053] One skilled in the art is aware of various references
including lists of cosmetic raw materials which may be used in
personal care compositions. Two such references are CTFA
International Buyers' Guide, 2002, Cosmetic, Toiletry and Fragrance
Association, Wash. D.C. and CTFA International Cosmetic Ingredient
Dictionary and Handbook, 7th Ed. (1997) Vol. 2, Eds. Wenninger and
McEwen, Cosmetic, Toiletry and Fragrance Association, Wash. D.C.
Also reference is made to WO 00/24372; WO 96/16630 and Sagarin,
Cosmetics, Science and Technology, 2nd Ed. Vol. 1 (1972).
[0054] Accordingly, the following examples are offered by way of
illustration, and are not meant to limit the invention in any
manner. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein.
[0055] The contents of all references, patents and published patent
applications cited throughout this application are hereby
incorporated by reference in their entirety.
Experimental
[0056] The procedures for restriction digest, ligation, preparation
of competent cells using calcium chloride, preparation of 20 mg/ml
isopropyl (IPTG), preparation of 20 mg/ml
5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside (X-gal), and
preparation of phosphate-buffered saline (PBS) were according to
well-known methods in the art and can be found in Sambrook et al.
(1989) supra. Phage-displayed libraries (cyclic 7-mer, linear 7-mer
and linear 12-mer) were supplied by New England Biolabs ((NEB;
Beverly, Mass.). Restriction endonucleases Eagl and Acc651,
10.times. NEBuffer 3, T4 DNA ligase, alkaline calf intestinal
phosphatase, E. coli ER2537 host strain, and M13KE gill cloning
vector were supplied by NEB and used according to the
manufacturer's instructions unless stated otherwise. Taq
polymerase, 10.times. PCR Buffer, and dNTP mix were supplied by
Roche Molecular Biochemicals (Indianapolis, Ind.). The HotStart Taq
Master Mix kit for PCR came from Qiagen (Valencia, Calif.). PCR was
carried out using a HYBAID Omn-E Thermocycler from E&K
Scientific Products (Campbell, Calif.) or PTC 2000 DNA Engine.TM.
from M. J. Research Inc. (Roche Molecular Systems, Inc. Alameda,
Calif.). Nondenaturing polyacrylamide gels (8%) and D-15 DNA
markers were obtained from Novex (San Diego, Calif.) and 2% E-gels
and TOPO cloning kits were obtained from Invitrogen (Carlsbad,
Calif.). Both the QIAquick Gel Extraction Kit and QIAquick PCR
Purification Kit were obtained from QIAGEN (Valencia, Calif.).
AmpliWax.TM. PCR Gems were obtained from Perkin Elmer (Boston,
Mass.).
EXAMPLE 1
Screening for Peptides Selected to Target Human Skin and not
Hair.
[0057] Two 3 inch strands of dark human hair (International Hair
Importers & Products, White Plains, N.Y.) were placed in BSA
blocked 50 ml conical tubes containing 10 ml of a 2%
Neutrogena.RTM. Bath Gel (Neutrogena Corp.) solution in Dl water.
10 .mu.L of cyclic 7-mer or linear 12-mer peptide libraries
(10.sup.10 pfu/.mu.l), or wild type phage (10.sup.9 pfu/.mu.l) were
added and the samples mixed at room temperature for 15 min with
rotatory shaking (30 rpm). The unbound supernatant was transferred
to a new tube containing an additional two 3 inch strands of dark
hair, and incubated at room temperature for 15 min with rotary
shaking. After this second hair incubation, 500 .mu.l of the
solution was transferred to the surface of human skin tissues
(EpiDerm.TM., MatTek Corp. Ashland, Mass.) in a 6 well culture
plate containing 0.9 mL tissue culture media (MatTek Corp) for 30
minutes at room temperature with gentle agitation. The skin tissues
were removed and washed 2.times. in 50 mls of 2% bath gel for 5 min
each and 3.times. in 50 mis of PBS for 5 min each in blocked 50 mL
conical tubes. After the final PBS wash, the skin tissues were
frozen at -20.degree. C. followed by PCR of the target bound ligand
phage. Table 1 illustrates the target bound skin peptides screened
according to this example.
EXAMPLE 2
Screening for Peptides Selected to Target Human Hair and Not
Skin.
[0058] Pre-equilibrated skin tissues were placed into a 6 well
culture plate containing fresh 0.9 mL tissue culture media and 300
.mu.l of a 2% Neutrogena.RTM. Bath Gel containing, 10 .mu.L of
cyclic 7-mer or linear 12-mer peptide libraries (10.sup.10
pfu/.mu.l), or wild type phage (10.sup.9 pfu/.mu.l) were added to
the skin surface. The samples were incubated at room temperature
for 15 min with gentle agitation. The unbound supernatant was
transferred to a new well containing skin tissue and the procedure
was repeated. The incubation solution was transferred to nine 3
inch dark hair (International Hair Importers & Products, White
Plains, N.Y.) strands in 50 ml tubes containing 10 ml of 2% body
gel for 30 minutes at room temperature with rotatory shaking (30
rpm). The hair samples were then washed with 1.times.50 mls,
2.times.50 mls, or 4.times.50 mls of 2% bath gel; Wash cycles in
PBS followed (1.times.25 mls for 5 min, 1.times.25 mls for 2 min,
2.times.50 mls for 5 min each, 150 mls total). After the final PBS
wash, the hair samples containing bound phage peptides were frozen
at -20.degree. C. Table 2 illustrates target bound hair peptides
screened according to this example.
EXAMPLE 3
Selection of Phage-Peptides that Bind to Hair or Skin Using PCR for
Identification of High Affinity Phage-Peptide Clones.
[0059] The skin swatches and hair samples were frozen at
-20.degree. C. until PCR. In one example, PCR was performed
directly on the hair and skin samples using the following
conditions in 0.5 ml PCR tubes with the following reagents: [0060]
50 .mu.l reaction mix (HotStart) [0061] 2 .mu.l CB05 primer (25
.mu.M) [0062] 2 .mu.l CB12 primer (25 .mu.M) [0063] 46 .mu.l
sterile dH.sub.2O
[0064] 5 .mu.l of BSA at 10 mg/ml and 50 .mu.l of mineral oil were
added. PCR amplification was performed post initiation at
95.degree. C. for 15 min, using 30 cycles of denaturation at
94.degree. C. for 30 sec, annealing at 58.degree. C. for 30 sec and
synthesis at 72.degree. C. for 60 sec. Extension was preformed at
72.degree. C. for 10 min. Primers were obtained from Operon
Technologies, Inc. (Alameda, Calif.). The sequences of the primers
were TABLE-US-00005 SEQ ID NO. 63 CB05
CGTAGTGGCATTACGTATTTTACCCGTTTAATGG (5' - 3') SEQ ID NO. 64 CB12
CGAGAGGGTTGATATAAGTATAGCCCGGAATAGG (5' - 3')
Additionally 1 .mu.l of the different PCR products was subjected to
another round of PCR using the same program but the following
ingredients were added: [0065] 50 .mu.l reaction mix (HotStart)
[0066] 1 .mu.l CM13 01 primer (50 .mu.M) [0067] 1 .mu.l CM13 02
primer (50 .mu.M) [0068] 47 .mu.l sterile dH.sub.2O
[0069] 50 .mu.l of BSA at 10 mg/ml and .mu.l of mineral oil were
added. Primers were obtained from Operon Technologies, Inc.
(Alameda, Calif.). The sequences for the primers were
TABLE-US-00006 SEQ ID NO. 65 CM13 01 CCTCGAAAGCAAGCTGATAAACCG
(5'-3') SEQ ID NO. 66 CM13 02 CATTCCACAGACAACCCTCATAG. (5'-3')
[0070] The PCR products were visualized on a 2% E-gel along with
PCR products from dilutions of the various initial phage peptide
libraries (positive control) and molecular weight markers, run
under 65V for 40 min. 4 .mu.l of the PCR products were subject to
TOPO cloning and transformation according to standard protocol but
all incubations were done for 30 minutes. The individual clones
were submitted to PCR (12.5 .mu.l Master Mix, 0.1 .mu.l each of
CM13 01 and CM13 02 primers, 12.3 .mu.l sterile water per clone)
using the same program as described above. Sequencing using 1 .mu.l
of PCR product and 11 .mu.l of g96 primer was completed at
Sequetech (Mountain View, Calif.); Biotech Core, Inc (Mountain
View, Calif.) or internally using an ABI Applied Biosystem
373XL.
EXAMPLE 4
Cloning of PCR Products.
[0071] PCR products from the first round of selection were cloned
as follows: Vector preparation:
[0072] 10 .mu.g of M13KE vector (New England Biolabs (NEB),
Beverly, Mass.) was digested overnight (16 h) at 37.degree. C. and
according to NEB recommended conditions, digestion was performed in
400 .mu.l total volume as follows: M13KE, 10 .mu.l; Eag 1, 10
.mu.l; Acc65 I, 10 .mu.l; 10.times.NEB buffer 3, 40 .mu.l;
100.times.BSA 4 .mu.l; and dH.sub.2O, 326 .mu.l. The digested
vector was purified using Qiagen PCR Purification Kit (Qiagen)
using 30 .mu.l of elution buffer (EB). The purified digest was
stored at -20.degree. C.
Insert Preparation:
[0073] PCR product from the first round of selection was purified
using the Qiagen Purification Kit and eluted in 30 .mu.l of EB
buffer. 15 .mu.l of the purified material was digested overnight in
100 .mu.l total volume as follows: PCR product, 15 .mu.l; Eag1, 1
.mu.l; Acc651, 1 .mu.l; 10.times.NEB buffer 3, 10 .mu.l;
100.times.BSA, 1 .mu.l; and dH.sub.2O, 64 .mu.l. The digestion was
followed by a heat shock treatment at 60.degree. C. for 20 min and
the product was stored at -20.degree. C. until further use.
[0074] The ligation was performed as described below using the
Takara kit at 16.degree. C. for 30 min, then placed on ice. Vector
preparation, 1 .mu.l; Insert preparation, 1 .mu.l; EB buffer, 3
.mu.l; and Solution 1, 5 .mu.l from Takara Biolnc., (Shiga,
Japan).
Transformation:
[0075] 5 .mu.l of ligation mixture was used to transform 50 .mu.l
of TOP10F' chemically competent cells (Invitrogen) according to the
commercial protocol. The cells were grown on LB plates overnight at
37.degree. C.
[0076] The phage peptide libraries were amplified and titered
according to standard techniques. Subsequent rounds of deselection
and selection may also be performed according to the methods
described above.
EXAMPLE 5
Stability of Phage-Peptide Libraries in Shampoo and Bath Gel
[0077] Stability of the three phage display libraries (Ph.D.-7,
Ph.D.-C7C and Ph.D.-12, New England Biolabs) was evaluated in a 2%
solution of commercially available Neutrogena.RTM. Anti-Residue
shampoo and in a 2% solution of Neutrogena.RTM. Bath Gel. 10 .mu.L
of each phage display library was added to 150 .mu.L of either the
shampoo or the bath gel solution in a micro titer plate (MTP).
After 30 minutes, 60 minutes and 120 minutes, a 20 .mu.L aliquot
was removed from each well and serially diluted in 180 .mu.L of LB
broth. The diluted samples, containing the phage peptide libraries,
from 10.sup.6 to 10.sup.4 PFU/mL, were added to 20 .mu.L of 20
mg/mL isopropyl-.beta.-D-thiogalactopyranoside (IPTG) and 200 .mu.L
of E. coli ER2537 cell culture in LB broth, mixed and incubated for
1 to 5 minutes. The infected cells were added to 3 mL of pre-heated
(55.degree. C.) LB agar tops containing 20 .mu.L of 40 mg/mL X-gal
(in DSMO), vortexed and immediately poured over pre-heated
(37.degree. C.) LA agar plates. Plates were cooled and incubated
overnight at 37.degree. C. The number of colonies on each plate
were counted and the number of plaque forming units per mL (pfu/mL)
were calculated for each plate. pfu/mL=(# colonies/10 .mu.L
phage).times.(dilution factor).times.(1000 .mu.L/1 mL)
[0078] FIGS. 2 and 3 illustrate the stability of the phage
populations in shampoo (FIG. 2) and shower gel (FIG. 3). The phage
display libraries were more stable in the bath gel solution than in
the shampoo solution. The pfu/mL of the phage libraries decreased
by less than one log unit in the bath gel solution, but they
decreased by up to two log units in the shampoo.
Sequence CWU 1
1
111 1 12 PRT Artificial Sequence synthetic skin binding peptide 1
Lys Gln Ala Thr Phe Pro Pro Asn Pro Thr Ala Tyr 1 5 10 2 7 PRT
Artificial Sequence synthetic skin binding peptide 2 Gln Ala Thr
Phe Met Tyr Asn 1 5 3 12 PRT Artificial Sequence synthetic skin
binding peptide 3 His Gly His Met Val Ser Thr Ser Gln Leu Ser Ile 1
5 10 4 11 PRT Artificial Sequence synthetic skin binding peptide 4
Val Leu Thr Ser Gln Leu Pro Asn His Ser Met 1 5 10 5 7 PRT
Artificial Sequence synthetic skin binding peptide 5 Leu Ser Pro
Ser Arg Met Lys 1 5 6 7 PRT Artificial Sequence synthetic skin
binding peptide 6 Leu Pro Ile Pro Arg Met Lys 1 5 7 7 PRT
Artificial Sequence synthetic skin binding peptide 7 His Ser Thr
Ala Tyr Leu Thr 1 5 8 7 PRT Artificial Sequence synthetic skin
binding peptide 8 His Gln Arg Pro Tyr Leu Thr 1 5 9 12 PRT
Artificial Sequence synthetic skin binding peptide 9 Ala Pro Gln
Gln Arg Pro Met Lys Thr Phe Asn Thr 1 5 10 10 12 PRT Artificial
Sequence synthetic skin binding peptide 10 Ala Pro Gln Gln Arg Pro
Met Lys Thr Val Gln Tyr 1 5 10 11 7 PRT Artificial Sequence
synthetic skin binding peptide 11 Pro Pro Trp Leu Asp Leu Leu 1 5
12 7 PRT Artificial Sequence synthetic skin binding peptide 12 Pro
Pro Trp Thr Phe Pro Leu 1 5 13 7 PRT Artificial Sequence synthetic
skin binding peptide 13 Ser Val Thr His Leu Thr Ser 1 5 14 7 PRT
Artificial Sequence synthetic skin binding peptide 14 Val Ile Thr
Arg Leu Thr Ser 1 5 15 7 PRT Artificial Sequence synthetic skin
binding peptide 15 Phe Pro Pro Leu Leu Arg Leu 1 5 16 12 PRT
Artificial Sequence synthetic skin binding peptide 16 Asp Leu Lys
Pro Pro Leu Leu Ala Leu Ser Lys Val 1 5 10 17 12 PRT Artificial
Sequence synthetic skin binding peptide 17 Ser His Pro Ser Gly Ala
Leu Gln Glu Gly Thr Phe 1 5 10 18 12 PRT Artificial Sequence
synthetic skin binding peptide 18 Phe Pro Leu Thr Ser Lys Pro Ser
Gly Ala Cys Thr 1 5 10 19 12 PRT Artificial Sequence synthetic skin
binding peptide 19 Asp Leu Lys Pro Pro Leu Leu Ala Leu Ser Lys Val
1 5 10 20 7 PRT Artificial Sequence synthetic skin binding peptide
20 Pro Leu Leu Ala Leu His Ser 1 5 21 7 PRT Artificial Sequence
synthetic skin binding peptide 21 Tyr Pro Ile Ser Thr Gln Ile 1 5
22 12 PRT Artificial Sequence synthetic skin binding peptide 22 Tyr
Ala Lys Gln His Tyr Pro Ile Ser Thr Phe Lys 1 5 10 23 7 PRT
Artificial Sequence synthetic skin binding peptide 23 His Ser Thr
Ala Tyr Leu Thr 1 5 24 12 PRT Artificial Sequence synthetic skin
binding peptide 24 Ser Thr Ala Tyr Leu Val Ala Met Ser Ala Ala Pro
1 5 10 25 12 PRT Artificial Sequence synthetic skin binding cluster
sequence 25 Ala Pro Gln Gln Arg Pro Met Xaa Thr Xaa Xaa Xaa 1 5 10
26 7 PRT Artificial Sequence synthetic skin binding cluster
sequence 26 Pro Pro Trp Xaa Xaa Xaa Leu 1 5 27 7 PRT Artificial
Sequence synthetic skin binding cluster sequence 27 Xaa Xaa Thr Xaa
Leu Thr Ser 1 5 28 7 PRT Artificial Sequence synthetic skin binding
cluster sequence 28 Xaa Pro Pro Leu Leu Xaa Leu 1 5 29 7 PRT
Artificial Sequence synthetic skin binding cluster sequence 29 Ser
Xaa Pro Ser Gly Ala Xaa 1 5 30 12 PRT Artificial Sequence synthetic
skin binding cluster sequence 30 Xaa Gln Ala Thr Phe Xaa Xaa Asn
Xaa Xaa Xaa Xaa 1 5 10 31 12 PRT Artificial Sequence synthetic skin
binding cluster sequence 31 Val Xaa Thr Ser Gln Leu Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 32 7 PRT Artificial Sequence synthetic skin binding
cluster sequence 32 Leu Xaa Xaa Xaa Arg Met Lys 1 5 33 7 PRT
Artificial Sequence synthetic skin binding cluster sequence 33 His
Xaa Xaa Xaa Tyr Leu Thr 1 5 34 7 PRT Artificial Sequence synthetic
hair binding peptide 34 Asn Thr Pro Lys Glu Asn Trp 1 5 35 7 PRT
Artificial Sequence synthetic hair binding peptide 35 Asn Thr Pro
Ala Ser Asn Arg 1 5 36 7 PRT Artificial Sequence synthetic hair
binding peptide 36 Pro Arg Gly Met Leu Ser Thr 1 5 37 7 PRT
Artificial Sequence synthetic hair binding peptide 37 Pro Pro Thr
Tyr Leu Ser Thr 1 5 38 12 PRT Artificial Sequence synthetic hair
binding peptide 38 Thr Ile Pro Thr His Arg Gln His Asp Tyr Arg Ser
1 5 10 39 7 PRT Artificial Sequence synthetic hair binding peptide
39 Thr Pro Pro Thr His Arg Leu 1 5 40 7 PRT Artificial Sequence
synthetic hair binding peptide 40 Leu Pro Thr Met Ser Thr Pro 1 5
41 7 PRT Artificial Sequence synthetic hair binding peptide 41 Leu
Gly Thr Asn Ser Thr Pro 1 5 42 12 PRT Artificial Sequence synthetic
hair binding peptide 42 Thr Pro Leu Thr Gly Ser Thr Asn Leu Leu Ser
Ser 1 5 10 43 7 PRT Artificial Sequence synthetic hair binding
peptide 43 Thr Pro Leu Thr Lys Glu Thr 1 5 44 7 PRT Artificial
Sequence synthetic hair binding peptide 44 Lys Gln Ser His Asn Pro
Pro 1 5 45 7 PRT Artificial Sequence synthetic hair binding peptide
45 Gln Gln Ser His Asn Pro Pro 1 5 46 7 PRT Artificial Sequence
synthetic hair binding peptide 46 Thr Gln Pro His Asn Pro Pro 1 5
47 12 PRT Artificial Sequence synthetic hair binding peptide 47 Ser
Thr Asn Leu Leu Arg Thr Ser Thr Val His Pro 1 5 10 48 12 PRT
Artificial Sequence synthetic hair binding peptide 48 His Thr Gln
Pro Ser Tyr Ser Ser Thr Asn Leu Phe 1 5 10 49 7 PRT Artificial
Sequence synthetic hair binding peptide 49 Ser Leu Leu Ser Ser His
Ala 1 5 50 12 PRT Artificial Sequence synthetic hair binding
peptide 50 Gln Gln Ser Ser Ile Ser Leu Ser Ser His Ala Val 1 5 10
51 7 PRT Artificial Sequence synthetic hair binding peptide 51 Asn
Ala Ser Pro Ser Ser Leu 1 5 52 7 PRT Artificial Sequence synthetic
hair binding peptide 52 His Ser Pro Ser Ser Leu Arg 1 5 53 7 PRT
Artificial Sequence synthetic hair binding peptide 53 Lys Xaa Ser
His His Thr His 1 5 54 7 PRT Artificial Sequence synthetic hair
binding peptide 54 Glu Xaa Ser His His Thr His 1 5 55 12 PRT
Artificial Sequence synthetic hair binding peptide 55 Ser His His
Thr His Tyr Gly Gln Pro Gly Pro Val 1 5 10 56 7 PRT Artificial
Sequence synthetic hair binding peptide 56 Leu Glu Ser Thr Ser Leu
Leu 1 5 57 7 PRT Artificial Sequence synthetic hair binding cluster
sequence 57 Asn Thr Pro Xaa Xaa Asn Xaa 1 5 58 7 PRT Artificial
Sequence synthetic hair binding cluster sequence 58 Pro Xaa Xaa Xaa
Leu Ser Thr 1 5 59 6 PRT Artificial Sequence synthetic hair binding
cluster sequence 59 Thr Xaa Pro Thr His Arg 1 5 60 7 PRT Artificial
Sequence synthetic hair binding cluster sequence 60 Leu Xaa Thr Xaa
Ser Thr Pro 1 5 61 7 PRT Artificial Sequence synthetic hair binding
cluster sequence 61 Thr Pro Leu Thr Xaa Xaa Thr 1 5 62 7 PRT
Artificial Sequence synthetic hair binding cluster sequence 62 Xaa
Gln Xaa His Asn Pro Pro 1 5 63 34 DNA Artificial Sequence primer 63
cgtagtggca ttacgtattt tacccgttta atgg 34 64 34 DNA Artificial
Sequence primer 64 cgagagggtt gatataagta tagcccggaa tagg 34 65 24
DNA Artificial Sequence primer 65 cctcgaaagc aagctgataa accg 24 66
23 DNA Artificial Sequence primer 66 cattccacag acaaccctca tag 23
67 4 PRT Artificial Sequence synthetic skin binding peptide
repeatable motif 67 Gln Ala Thr Phe 1 68 4 PRT Artificial Sequence
synthetic skin binding peptide repeatable motif 68 Thr Ser Gln Leu
1 69 7 PRT Artificial Sequence synthetic skin binding peptide
repeatable motif 69 Ala Pro Gln Gln Arg Pro Met 1 5 70 4 PRT
Artificial Sequence synthetic skin binding peptide repeatable motif
70 Pro Met Lys Thr 1 71 4 PRT Artificial Sequence synthetic skin
binding peptide repeatable motif 71 Pro Pro Leu Leu 1 72 8 PRT
Artificial Sequence synthetic skin binding peptide repeatable motif
72 Ala Pro Gln Gln Arg Met Lys Thr 1 5 73 4 PRT Artificial Sequence
synthetic skin binding peptide repeatable motif 73 Pro Ser Gly Ala
1 74 5 PRT Artificial Sequence synthetic skin binding peptide
repeatable motif 74 Pro Leu Leu Ala Leu 1 5 75 5 PRT Artificial
Sequence synthetic skin binding peptide repeatable motif 75 Ser Thr
Ala Tyr Leu 1 5 76 5 PRT Artificial Sequence synthetic skin binding
peptide repeatable motif 76 Tyr Pro Ile Ser Thr 1 5 77 4 PRT
Artificial Sequence synthetic hair binding peptide repeatable motif
77 Ser Thr Asn Leu 1 78 5 PRT Artificial Sequence synthetic hair
binding peptide repeatable motif 78 Leu Ser Ser His Ala 1 5 79 5
PRT Artificial Sequence synthetic hair binding peptide repeatable
motif 79 Ser Pro Ser Ser Leu 1 5 80 5 PRT Artificial Sequence
synthetic hair binding peptide repeatable motif 80 Ser His His Thr
His 1 5 81 4 PRT Artificial Sequence synthetic hair binding peptide
repeatable motif 81 Pro Thr His Arg 1 82 4 PRT Artificial Sequence
synthetic hair binding peptide repeatable motif 82 Thr Pro Leu Thr
1 83 4 PRT Artificial Sequence synthetic hair binding peptide
repeatable motif 83 His Asn Pro Pro 1 84 9 PRT Artificial Sequence
synthetic skin binding peptide derivative 84 Cys Gln Ala Thr Phe
Met Tyr Asn Cys 1 5 85 9 PRT Artificial Sequence synthetic skin
binding peptide derivative 85 Cys Leu Ser Pro Ser Arg Met Lys Cys 1
5 86 9 PRT Artificial Sequence synthetic skin binding peptide
derivative 86 Cys Leu Pro Ile Pro Arg Met Lys Cys 1 5 87 9 PRT
Artificial Sequence synthetic skin binding peptide derivative 87
Cys His Ser Thr Ala Tyr Leu Thr Cys 1 5 88 9 PRT Artificial
Sequence synthetic skin binding peptide derivative 88 Cys His Gln
Arg Pro Tyr Leu Thr Cys 1 5 89 9 PRT Artificial Sequence synthetic
skin binding peptide derivative 89 Cys Pro Pro Trp Leu Asp Leu Leu
Cys 1 5 90 9 PRT Artificial Sequence synthetic skin binding peptide
derivative 90 Cys Pro Pro Trp Leu Asp Leu Leu Cys 1 5 91 9 PRT
Artificial Sequence synthetic skin binding peptide derivative 91
Cys Ser Val Thr His Leu Thr Ser Cys 1 5 92 9 PRT Artificial
Sequence synthetic skin binding peptide derivative 92 Cys Val Ile
Thr Arg Leu Thr Ser Cys 1 5 93 9 PRT Artificial Sequence synthetic
skin binding peptide derivative 93 Cys Phe Pro Pro Leu Leu Arg Leu
Cys 1 5 94 9 PRT Artificial Sequence synthetic skin binding peptide
derivative 94 Cys Pro Leu Leu Ala Leu His Ser Cys 1 5 95 9 PRT
Artificial Sequence synthetic skin binding peptide derivative 95
Cys Tyr Pro Ile Ser Thr Gln Ile Cys 1 5 96 9 PRT Artificial
Sequence synthetic skin binding peptide derivative 96 Cys His Ser
Thr Ala Tyr Leu Thr Cys 1 5 97 9 PRT Artificial Sequence synthetic
hair binding peptide derivative 97 Cys Asn Thr Pro Lys Glu Asn Trp
Cys 1 5 98 9 PRT Artificial Sequence synthetic hair binding peptide
derivative 98 Cys Asn Thr Pro Ala Ser Asn Arg Cys 1 5 99 9 PRT
Artificial Sequence synthetic hair binding peptide derivative 99
Cys Pro Arg Gly Met Leu Ser Thr Cys 1 5 100 9 PRT Artificial
Sequence synthetic hair binding peptide derivative 100 Cys Pro Pro
Thr Tyr Leu Ser Thr Cys 1 5 101 9 PRT Artificial Sequence synthetic
hair binding peptide derivative 101 Cys Thr Pro Pro Thr His Arg Leu
Cys 1 5 102 9 PRT Artificial Sequence synthetic hair binding
peptide derivative 102 Cys Leu Pro Thr Met Ser Thr Pro Cys 1 5 103
9 PRT Artificial Sequence synthetic hair binding peptide derivative
103 Cys Leu Gly Thr Asn Ser Thr Pro Cys 1 5 104 9 PRT Artificial
Sequence synthetic hair binding peptide derivative 104 Cys Thr Pro
Leu Thr Lys Glu Thr Cys 1 5 105 9 PRT Artificial Sequence synthetic
hair binding peptide derivative 105 Cys Thr Gln Pro His Asn Pro Pro
Cys 1 5 106 9 PRT Artificial Sequence synthetic hair binding
peptide derivative 106 Cys Ser Leu Leu Ser Ser His Ala Cys 1 5 107
9 PRT Artificial Sequence synthetic hair binding peptide derivative
107 Cys Asn Ala Ser Pro Ser Ser Leu Cys 1 5 108 9 PRT Artificial
Sequence synthetic hair binding peptide derivative 108 Cys His Ser
Pro Ser Ser Leu Arg Cys 1 5 109 9 PRT Artificial Sequence synthetic
hair binding peptide derivative 109 Cys Leu Glu Ser Thr Ser Leu Leu
Cys 1 5 110 4 PRT Artificial Sequence synthetic linker 110 Gly Gly
Gly Lys 1 111 5 PRT Artificial Sequence synthetic linker 111 Gly
Gly His Gly Gly 1 5
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