U.S. patent application number 11/778699 was filed with the patent office on 2007-11-22 for peptide-based body surface coloring reagents.
Invention is credited to John P. O'Brien, Hong Wang, Ying Wu.
Application Number | 20070269394 11/778699 |
Document ID | / |
Family ID | 38445831 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269394 |
Kind Code |
A1 |
O'Brien; John P. ; et
al. |
November 22, 2007 |
PEPTIDE-BASED BODY SURFACE COLORING REAGENTS
Abstract
Peptides have been identified that bind with high affinity to
body surfaces, such as, hair, skin, nails, teeth, gums, and oral
cavity surfaces. Diblock and triblock peptide-based body surface
coloring reagents formed by coupling a body surface binding peptide
to a pigment binding peptide, either directly or through a spacer,
are described. The peptide-based body surface coloring reagents may
be used in conjunction with pigments to color body surfaces.
Inventors: |
O'Brien; John P.; (Oxford,
PA) ; Wang; Hong; (Kennett Square, PA) ; Wu;
Ying; (Wallingford, PA) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
38445831 |
Appl. No.: |
11/778699 |
Filed: |
July 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11389948 |
Mar 27, 2006 |
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11778699 |
Jul 17, 2007 |
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11074473 |
Mar 8, 2005 |
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11389948 |
Mar 27, 2006 |
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10935642 |
Sep 7, 2004 |
7220405 |
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11074473 |
Mar 8, 2005 |
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60501498 |
Sep 8, 2003 |
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Current U.S.
Class: |
424/64 ; 424/63;
435/69.1; 525/54.1 |
Current CPC
Class: |
A61Q 3/00 20130101; A61K
2800/57 20130101; A61Q 1/02 20130101; A61Q 19/04 20130101; A61K
8/64 20130101; A61K 2800/42 20130101; A61Q 5/065 20130101; A61K
2800/94 20130101; A61Q 11/00 20130101; C07K 7/06 20130101; C07K
7/08 20130101 |
Class at
Publication: |
424/064 ;
424/063; 525/054.1; 435/069.1 |
International
Class: |
A61K 8/64 20060101
A61K008/64; C12P 21/06 20060101 C12P021/06; C08G 63/91 20060101
C08G063/91 |
Claims
1. A diblock, peptide-based body surface coloring reagent having
the general structure [(BSBP).sub.m-(PBP).sub.n].sub.x, wherein a)
BSBP is a body surface binding peptide; b) PBP is a pigment-binding
peptide; and c) m, n, and x independently range from 1 to about
10.
2. A triblock, peptide-based body surface coloring reagent having
the general structure
[[(BSBP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z]y,
wherein a) BSBP is a body surface binding peptide; b) PBP is a
pigment-binding peptide; c) S is a molecular spacer; and d) m, n, x
and z independently range from 1 to about 10, y is from 1 to about
5, and where q and r are each independently 0 or 1, provided that
both r and q may not be 0.
3. A peptide-based body surface coloring reagent according to claim
1 or 2 wherein either one of or both the body surface binding
peptide and the pigment-binding peptide is generated
combinatorially by a process selected from the group consisting of
phage display, yeast display, bacteria display and combinatorial
solid phase peptide synthesis.
4. A peptide-based body surface coloring reagent according to claim
3 wherein the body surface binding peptide is from about 7 to about
50 amino acids.
5. A peptide-based body surface coloring reagent according to claim
1 or 2 wherein the body surface binding peptide is generated
empirically.
6. A peptide-based body surface coloring reagent according to claim
1 or 2 wherein the body surface binding peptide binds to body
surfaces selected from the group consisting of, nails, teeth, gums,
skin, and tissues of the oral cavity.
7. A peptide-based body surface coloring reagent according to claim
6 wherein the body surface-binding peptide is from about 7 to about
25 amino acids and has a binding affinity for a body surface,
measured as MB.sub.50, equal to or less than 10.sup.-5 M.
8. A peptide-based body surface coloring reagent according to claim
6 wherein the body surface comprises an epithelial cell layer.
9. A peptide-based body surface coloring reagent according to claim
8 wherein the body surface comprises an endothelial cell layer.
10. A peptide-based body surface coloring reagent according to
claim 1 or wherein the body surface binding peptide is a
hair-binding peptide selected from the group consisting of SEQ ID
NOs:1, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 58, 59, 60, 64,
66, 69, 70, 75, 105, 106, 107, 108, 109, 153, 154, 155, and
156.
11. A peptide-based body surface coloring reagent according to
claim 1 or wherein the body surface binding peptide is a
skin-binding peptide selected from the group consisting of SEQ ID
NOs:2, 61, 99, 100, 101, 102, 103, and 104.
12. A peptide-based body surface coloring reagent according to
claim 1 or 2 wherein the body surface binding peptide is a
nail-binding peptide selected from the group consisting of SEQ ID
NO: SEQ ID NOs:7, 8, 19-27, 38-40, 43, 44, 47, 53, 57, 58, 59 and
60.
13. A peptide-based body surface coloring reagent according to
claim 1 or 2 wherein the pigment-binding peptide is from about 5 to
about 50 amino acids.
14. A peptide-based body surface coloring reagent according to
claim 1 or wherein the pigment-binding peptide has affinity for a
pigment selected from the group consisting of D&C Red No. 36,
D&C Red No. 30, D&C Orange No. 17, Green 3 Lake, Ext.
Yellow 7 Lake, Orange 4 Lake, Red 28 Lake; the calcium lakes of
D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&C Red
No. 12, the strontium lake D&C Red No. 13, the aluminum lakes
of FD&C Yellow No. 5, of FD&C Yellow No. 6, of FD&C No.
40, of D&C Red Nos. 21, 22, 27, and 28, of FD&C Blue No. 1,
of D&C Orange No. 5, of D&C Yellow No. 10; the zirconium
lake of D&C Red No. 33; Cromophthal.RTM. Yellow, Sunfast.RTM.
Magenta, Sunfast.RTM. Blue, iron oxides, calcium carbonate,
aluminum hydroxide, calcium sulfate, kaolin, ferric ammonium
ferrocyamide, magnesium carbonate, carmine, barium sulfate, mica,
bismuth oxychloride, zinc stearate, manganese violet, chromium
oxide, titanium dioxide, titanium dioxide nanoparticles, zinc
oxide, barium oxide, ultramarine blue, bismuth citrate,
hydroxyapatite, zirconium silicate, and carbon black particles.
15. A peptide-based body surface coloring reagent according to
claim 1 or 2 wherein the pigment-binding peptide is selected from
the group consisting of SEQ ID NOs:110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, and 134.
16. A triblock peptide-based body surface coloring reagent
according to claim 2 wherein the spacer is selected from the group
consisting of ethanolamine, ethylene glycol, polyethylene with a
chain length of 6 carbon atoms, polyethylene glycol with 3 to 6
repeating units, phenoxyethanol, propanolamide, butylene glycol,
butyleneglycolamide, propyl phenyl chains, ethyl alkyl chains,
propyl alkyl chains, hexyl alkyl chains, steryl alkyl chains, cetyl
alkyl chains, and palmitoyl alkyl chains.
17. A triblock peptide-based body surface coloring reagent
according to claim 2 wherein the spacer is a peptide comprising
from 1 to about 50 amino acids.
18. A triblock peptide-based body surface coloring reagent
according to claim 17 wherein the spacer comprises amino acids
selected from the group consisting of proline, lysine, glycine,
alanine, serine, and mixtures thereof.
19. A triblock peptide-based body surface coloring reagent
according to claim 17 wherein the spacer comprises peptide
sequences selected from the group consisting of SEQ ID NOs:65, 135,
136, and 137.
20. A peptide-based body surface coloring reagent according to
claim 1 or 2 wherein the peptide-based body surface coloring
reagent is from about 14 to about 200 amino acids.
21. A triblock peptide-based body surface coloring reagent
according to claim 2 wherein the triblock peptide-based body
coloring reagent has an amino acid sequence selected from the group
consisting of SEQ ID NOs:138, 139, 140, 141, 142, 143, 144, 145,
146, and 147.
22. A peptide-based body surface coloring reagent according to
claim 1 or 2 wherein the body surface-binding peptide is isolated
by a process comprising the steps of: (i) providing a library of
combinatorially generated phage-peptides; (ii) contacting the
library of (i) with a body surface to form a reaction solution
comprising: (A) phage-peptide-body surface complex; (B) unbound
body surface, and (C) uncomplexed peptides; (iii) isolating the
phage-peptide-body surface complex of (ii); (iv) eluting the weakly
bound peptides from the isolated peptide complex of (iii); (v)
identifying the remaining bound phage-peptides either by using the
polymerase chain reaction directly with the phage-peptide-body
surface complex remaining after step (iv), or by infecting
bacterial host cells directly with the phage-peptide-body surface
complex remaining after step (iv), growing the infected cells in a
suitable growth medium, and isolating and identifying the
phage-peptides from the grown cells.
23. A peptide-based body surface coloring reagent according to
claim 22 wherein the body surface is selected from the group
consisting of, nails, teeth, gums, skin, and tissues of the oral
cavity.
24. A personal care composition comprising an effective amount of
the peptide-based body surface coloring reagent of claim 1 or 2,
comprising a body surface binding peptide and a pigment binding
peptide.
25. A personal care composition according to claim 24 wherein; a)
the body surface binding peptide has affinity for a body surface
selected from the group consisting of, nails, teeth, gums, skin,
and tissues of the oral cavity; and b) the body surface binding
peptide is from about 7 to about 25 amino acids and has a binding
affinity for a body surface, measured as MB.sub.50, equal to or
less than 10.sup.-5M.
26. A method for coloring a body surface comprising: a) providing a
pigment; b) providing a composition comprising a peptide-based body
surface coloring reagent according to claim 1 or 2 wherein the body
surface binding peptide has affinity for the body surface and the
pigment binding peptide has affinity for the pigment; and c)
applying the pigment of (a) with the composition of (b) to a body
surface for a time sufficient for the peptide-based body surface
coloring reagent to bind to the pigment and the body surface.
27. A method according to claim 26 wherein the body surface is
selected from the group consisting of, nails, teeth, gums, skin,
and tissues of the oral cavity.
28. A method according to claim 26 wherein the pigment and the
composition comprising a peptide-based body surface coloring
reagent are applied to the body surface concomitantly.
29. A method according to claim 26 wherein the pigment is applied
to the body surface prior to the application of the composition
comprising a peptide-based body surface coloring reagent.
30. A method according to claim 26 wherein the composition
comprising a peptide based-body surface coloring reagent is applied
to the body surface prior to the application of the pigment.
31. A method according to claim 26 further comprising the step of:
d) applying a composition comprising a polymeric sealant to the
body surface subsequent to the applying of the pigment and the
composition comprising a peptide-based body surface coloring
reagent.
32. A method according to claim 31 wherein the polymeric sealant is
selected from the group consisting of poly(allylamine), acrylates,
acrylate copolymers, polyurethanes, carbomers, methicones,
amodimethicones, polyethylenene glycol, beeswax, and siloxanes.
Description
[0001] This patent application is a continuation in part of U.S.
patent application Ser. No. 11/074,473, filed Mar. 8, 2005, which
is a continuation in part of U.S. patent application Ser No.
10/935,642, filed Sep. 7, 2004, which claims the benefit of U.S.
Provisional Application 60/501,498, filed Sep. 8, 2003.
FIELD OF THE INVENTION
[0002] The invention relates to the field of personal care
products. More specifically, the invention relates to diblock and
triblock peptide-based body surface coloring reagents comprising
body surface-binding peptides and pigment-binding peptides that may
be used to attach pigments to body surfaces.
BACKGROUND OF THE INVENTION
[0003] Colorants for body surfaces, such as hair, skin, and nails
are well-known and frequently used in personal care products. Hair
coloring agents may be divided into three categories, specifically,
permanent, semi-permanent or direct, and temporary. The permanent
hair dyes are generally oxidative dyes that provide hair color that
lasts about four to six weeks. These oxidative hair dyes consist of
two parts, one part contains the oxidative dyes in addition to
other ingredients, while the second part contains an oxidizing
agent such as hydrogen peroxide. The two components are mixed
immediately prior to use. The oxidizing agent oxidizes the dye
precursors, which then combine to form large color molecules within
the hair shaft. Although the oxidative hair dyes provide
long-lasting color, the oxidizing agents they contain cause hair
damage. The semi-permanent or direct hair dyes are preformed dye
molecules that are applied to the hair and provide color for about
six to twelve shampoos. This type of hair dye is gentler to the
hair because it does not contain peroxides, but the hair color does
not last as long. Some improved durability is achieved by the use
of nanoparticle hair coloring materials with a particle size of 10
to 500 nm, as described by Hensen et al. in WO 01045652. These
nanoparticle hair coloring materials are conventional direct hair
dyes that are treated to obtain nanoscale dimensions and exhibit
increased absorption into the hair. Temporary hair dyes are
coloring agents that are applied to the hair surface and are
removed after one shampoo. It would be desirable to develop a hair
coloring agent that provides the durability of the permanent hair
dyes without the use of oxidizing agents that damage hair.
[0004] The major problem with the current skin colorants,
non-oxidative hair dyes, as well as nail coloring agents is that
they lack the required durability required for long-lasting
effects. For this reason, there have been attempts to enhance the
binding of cosmetic agents to the hair, skin or nails. For example,
Richardson et al. in U.S. Pat. No. 5,490,980 and Green et al. in
U.S. Pat. No. 6,267,957 describe the covalent attachment of
cosmetic agents, such as skin conditioners, hair conditioners,
coloring agents, sunscreens, and perfumes, to hair, skin, and nails
using the enzyme transglutaminase. This enzyme crosslinks an amine
moiety on the cosmetic agent to the glutamine residues in skin,
hair, and nails. Similarly, Green et al. in WO 0107009 describe the
use of the enzyme lysine oxidase to covalently attach cosmetic
agents to hair, skin, and nails.
[0005] In another approach, cosmetic agents have been covalently
attached to proteins or protein hydrolysates. For example, Lang et
al. in U.S. Pat. No. 5,192,332 describe temporary coloring
compositions that contain an animal or vegetable protein, or
hydrolysate thereof, which contain residues of dye molecules
grafted onto the protein chain. In those compositions, the protein
serves as a conditioning agent and does not enhance the binding of
the cosmetic agent to hair, skin, or nails. Horikoshi et al. in JP
08104614 and Igarashi et al. in U.S. Pat. No. 5,597,386 describe
hair coloring agents that consist of an anti-keratin antibody
covalently attached to a dye or pigment. The antibody binds to the
hair, thereby enhancing the binding of the hair coloring agent to
the hair. Similarly, Kizawa et al. in JP 09003100 describe an
antibody that recognizes the surface layer of hair and its use to
treat hair. A hair coloring agent consisting of that anti-hair
antibody coupled to colored latex particles is also described. The
use of antibodies to enhance the binding of dyes to the hair is
effective in increasing the durability of the hair coloring, but
these antibodies are difficult and expensive to produce. Terada et
al. in JP 2002363026 describe the use of conjugates consisting of
single-chain antibodies, preferably anti-keratin, coupled to dyes,
ligands, and cosmetic agents for skin and hair care compositions.
The single-chain antibodies may be prepared using genetic
engineering techniques, but are still difficult and expensive to
prepare because of their large size. Findlay in WO 00048558
describes the use of calycin proteins, such as
.beta.-lactoglobulin, which contain a binding domain for a cosmetic
agent and another binding domain that binds to at least a part of
the surface of a hair fiber or skin surface, for conditioners,
dyes, and perfumes. Again these proteins are large and difficult
and expensive to produce.
[0006] Linter in U.S. Pat. No. 6,620,419 describes peptides grafted
to a fatty acid chain and their use in cosmetic and
dermopharmaceutical applications. The peptides described in that
disclosure are chosen because they stimulate the synthesis of
collagen; they are not specific binding peptides that enhance the
durability of hair and skin conditioners, and hair, nail, and skin
colorants.
[0007] Peptide-based hair conditioners, hair colorants, and other
benefit agents have also been developed to improve the durability
of these compositions (Huang et al., copending and commonly owned
U.S. Patent Application Publication No. 2005/0050656, and U.S.
Patent Application Publication No. 2005/0226839). The peptide-based
hair conditioners or colorants are prepared by coupling a specific
peptide sequence that has a high binding affinity to hair with a
conditioning or coloring agent, respectively. The peptide portion
binds to the hair, thereby strongly attaching the conditioning or
coloring agent. Peptides with a high binding affinity to hair have
been identified using phage display screening techniques (Huang et
al., supra; Estell et al. WO 0179479; Murray et al., U.S. Patent
Application Publication No. 2002/0098524; Janssen et al., U.S.
Patent Application Publication No. 2003/0152976; and Janssen et
al., WO 04048399).
[0008] Additionally, Reisch (Chem. Eng. News 80:16-21 (2002))
reports that a family of peptides designed to target an ingredient
of specific human tissue has been developed for personal care
applications. However, no description of peptide-based conditioners
or coloring agents are disclosed in that publication. Although
these peptide-based reagents offer much promise in personal care
applications, they generally require covalent coupling of the
peptide to the coloring agent. The covalent coupling chemistry may
be complex and time consuming, and adds to the cost of the
reagent.
[0009] In view of the above, a need exists for colorants for body
surfaces, such as hair, skin, nails, and teeth that provide
improved durability for long lasting effects and are easy and
inexpensive to prepare.
[0010] Applicants have addressed the stated need by identifying
peptide sequences using phage display screening that specifically
bind to body surfaces, such as, hair, skin, nails, teeth, gums, and
oral cavity surfaces, with high affinity and coupling them with
specific pigment-binding peptides to provide diblock and triblock
peptide-based reagents that may be used in conjunction with
pigments to color body surfaces.
SUMMARY OF THE INVENTION
[0011] The invention provides peptide-based body surface coloring
reagents comprising a body surface binding peptide and a
pigment-binding peptide. These peptide-based reagents may be used
in conjunction with pigments to color body surfaces, such as hair,
skin, nails, and teeth. The body surface binding peptide binds
strongly to the body surface and the pigment-binding peptide binds
to the pigment, thereby attaching the pigment to the body
surface.
[0012] Accordingly, in one embodiment the invention provides a
diblock peptide-based body surface coloring having the general
structure [(BSBP).sub.m-(PBP).sub.n].sub.x, wherein [0013] a) BSBP
is a body surface binding peptide; [0014] b) PBP is a
pigment-binding peptide; and [0015] c) m, n, and x independently
range from 1 to about 10.
[0016] In another embodiment, the invention provides a triblock,
peptide-based body surface coloring reagent having the general
structure
[[(BSBP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z].sub.y,
wherein [0017] a) BSBP is a body surface binding peptide; [0018] b)
PBP is a pigment-binding peptide; [0019] c) S is a molecular
spacer; and [0020] d) m, n, x and z independently range from 1 to
about 10, y is from 1 to about 5, and where q and r are each
independently 0 or 1, provided that both r and q may not be 0.
[0021] In another embodiment the invention provides aq
peptide-based body surface coloring reagent according to the
invention wherein the body surface-binding peptide is isolated by a
process comprising the steps of: [0022] (i) providing a library of
combinatorially generated phage-peptides; [0023] (ii) contacting
the library of (i) with a body surface to form a reaction solution
comprising: [0024] (A) phage-peptide-body surface complex; [0025]
(B) unbound body surface, and [0026] (C) uncomplexed peptides;
[0027] (iii) isolating the phage-peptide-body surface complex of
(ii); [0028] (iv) eluting the weakly bound peptides from the
isolated peptide complex of (iii); [0029] (v) identifying the
remaining bound phage-peptides either by using the polymerase chain
reaction directly with the phage-peptide-body surface complex
remaining after step (iv), or by infecting bacterial host cells
directly with the phage-peptide-body surface complex remaining
after step (iv), growing the infected cells in a suitable growth
medium, and isolating and identifying the phage-peptides from the
grown cells.
[0030] In another embodiment the invention provides a personal care
composition comprising an effective amount of the peptide-based
body surface coloring reagent of the invention, comprising a body
surface binding peptide and a pigment binding peptide.
[0031] In a similar embodiment the invention provides a method for
coloring a body surface comprising: [0032] a) providing a pigment;
[0033] b) providing a composition comprising a peptide-based body
surface coloring reagent according to the invention wherein the
body surface binding peptide has affinity for the body surface and
the pigment binding peptide has affinity for the pigment; and
[0034] c) applying the pigment of (a) with the composition of (b)
to a body surface for a time sufficient for the peptide-based body
surface coloring reagent to bind to the pigment and the body
surface.
[0035] Additionally, the invention provides personal care
compositions, such as hair coloring, hair conditioning, skin
coloring, skin conditioning, cosmetic, oral care, and nail polish
compositions, comprising the peptide-based body surface coloring
reagents.
BRIEF DESCRIPTION OF FIGURES AND SEQUENCE DESCRIPTIONS
[0036] The invention can be more fully understood from the
following detailed description, figure and the accompanying
sequence descriptions, which form a part of this application.
[0037] FIG. 1 is a plasmid map of the vector pKSIC4-HC77623,
described in Examples 17-20.
[0038] The following sequences conform with 37 C.F.R. 1.821-1.825
("Requirements for Patent Applications Containing Nucleotide
Sequences and/or Amino Acid Sequence Disclosures--the Sequence
Rules") and consistent with World Intellectual Property
Organization (WIPO) Standard ST.25 (1998) and the sequence listing
requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and
Section 208 and Annex C of the Administrative Instructions). The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37C.F.R. .sctn.1.822.
[0039] A Sequence Listing is provided herewith on Compact Disk. The
contents of the Compact Disk containing the Sequence Listing are
hereby incorporated by reference in compliance with 37 CFR
1.52(e).
[0040] SEQ ID NO:1 is the amino acid sequence of a hair-binding
peptide.
[0041] SEQ ID NO:2 is the amino acid sequence of a skin-binding
peptide.
[0042] SEQ ID NOs:3-52, 54-59 are the amino acid sequences of
hair-binding peptides.
[0043] SEQ ID NO:53 is the amino acid sequence of a hair-binding
and nail-binding peptide.
[0044] SEQ ID NO:60 is the amino acid sequence of a nail-binding
peptide.
[0045] SEQ ID NO:61 is the amino acid sequence of a skin-binding
peptide.
[0046] SEQ ID NO:62 is the oligonucleotide primer used to sequence
phage DNA.
[0047] SEQ ID NO:63 is the amino acid sequence of a peptide used as
a control in the ELISA binding assay, as described in Example
5.
[0048] SEQ ID NO:64 is the amino acid sequence of a
cysteine-attached hair-binding peptide.
[0049] SEQ ID NO:65 is the amino acid sequence of the Caspase 3
cleavage site.
[0050] SEQ ID NOs:66, 69, and 70 are the amino acid sequence of
shampoo-resistant hair-binding peptides.
[0051] SEQ ID NOs:67 and 68 are the nucleotide sequences of the
primers used to amplify shampoo-resistant, hair-binding phage
peptides, as described in Example 8.
[0052] SEQ ID NOs:71-74 are the amino acid sequences of the
biotinylated hair-binding and skin-binding peptides used Example
9.
[0053] SEQ ID NO:75 is the amino acid sequence of a hair
conditioner resistant hair-binding peptide.
[0054] SEQ ID NOs:76-98 are the amino acid sequences of
hair-binding peptides.
[0055] SEQ ID NOs:99-104 are the amino acid sequences of
skin-binding peptides.
[0056] SEQ ID NOs:105-109 are the amino acid sequences of
empirically generated hair and skin-binding peptides.
[0057] SEQ ID NOs:110-134 are the amino acid sequences of
pigment-binding peptides.
[0058] SEQ ID NOs:135-137 are the amino acid sequences of peptide
spacers.
[0059] SEQ ID NO:138-147 are the amino acid sequences of triblock
peptide-based body surface coloring reagents.
[0060] SEQ ID NOs:148-151 are the nucleotide sequences that encode
the peptide-based body surface coloring reagents given as SEQ ID
NOs:144-147.
[0061] SEQ ID NO:152 is the nucleotide sequence of plasmid
pKSIC4-HCC77623, which is described in Examples 17-20.
[0062] SEQ ID NOs:153-156 are the amino acid sequences of hair
conditioner and shampoo resistant hair-binding peptides.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention provides diblock and triblock
peptide-based body surface coloring reagents which comprise at
least one body surface-binding peptide coupled to at least one
pigment-binding peptide, either directly or through a molecular
spacer. These diblock and triblock peptide-based reagents may be
used in conjunction with pigments to color body surfaces. Typical
of the compositions of the invention are peptide-based hair and
skin colorants and nail polish compositions.
[0064] The peptide based diblock and triblock peptide-based body
surface coloring reagents of the invention provide benefits and an
advance over the art in the development of personal care products.
Because the reagents are peptide based they are able to bind
strongly to body surfaces from an aqueous environment, thus in many
cases being both water soluble and water fast. Additionally,
because of the aqueous nature of the reagents, they may be removed
from body surfaces without of the use of odor producing chemicals.
The reagents of the invention bind almost immediately to the target
body surface, eliminating the need for long drying times, typical
of most personal care applications. Moreover, the peptide-based
body surface coloring reagents are used without the need to
covalently attach the body surface-binding peptide to the coloring
agent. Most importantly, the peptide nature of the reagents makes
them virtually non-toxic and non-irritating to exposed body
surfaces such as the skin and the membranes of the eyes and
mouth.
[0065] The following definitions are used herein and should be
referred to for interpretation of the claims and the
specification.
[0066] "HBP" means hair-binding peptide.
[0067] "SBP" means skin-binding peptide.
[0068] "NBP" means nail-binding peptide.
[0069] "OBP" means oral cavity surface-binding peptide.
[0070] "TBP" means tooth-binding peptide.
[0071] "PBP" means pigment-binding peptide.
[0072] "C" means coloring agent for body surfaces such as hair,
skin, nails, or teeth.
[0073] "S" means spacer.
[0074] "BSBP" means body surface binding peptide.
[0075] The term "present invention" or "invention" as used herein
is meant to apply generally to all embodiments of the invention as
recited in the claims as presented, or as later amended and
supplemented.
[0076] The term "peptide" refers to two or more amino acids joined
to each other by peptide bonds or modified peptide bonds.
[0077] The term "body surface" refers to any surface of the human
body that may serve as a substrate for the binding of a diblock or
triblock peptide-based body surface coloring reagent comprising at
least one body surface-binding peptide and at least one
pigment-binding peptide. Typical body surfaces include but are not
limited to hair, skin, nails, teeth, and gums.
[0078] The term "hair" as used herein refers to any type of human
hair, including eyebrows, eyelashes, and other facial hair.
[0079] The term "skin" as used herein refers to human skin, or
substitutes for human skin, such as pig skin, Vitro-Skin.RTM. and
EpiDerm.TM.. Skin as used herein as a body surface will generally
comprise a layer of epithelial cells and may additionally comprise
a layer of endothelial cells.
[0080] The term "nails" as used herein refers to human fingernails
and toenails.
[0081] The terms "coupling" and "coupled" as used herein refer to
any chemical association and includes both covalent and
non-covalent interactions.
[0082] The term "stringency" as it is applied to the selection of
the body-surface-binding peptides, refers to the concentration of
the eluting agent (usually detergent) used to elute peptides from
the body surface. Higher concentrations of the eluting agent
provide more stringent conditions.
[0083] The term "peptide-body surface complex" means structure
comprising a peptide bound to a sample of a body surface via a
binding site on the peptide.
[0084] The term "peptide-hair complex" means structure comprising a
peptide bound to a hair fiber via a binding site on the
peptide.
[0085] The term "peptide-skin complex" means structure comprising a
peptide bound to the skin via a binding site on the peptide.
[0086] The term "peptide-nail complex" means structure comprising a
peptide bound to fingernails or toenails via a binding site on the
peptide.
[0087] The term "peptide-substrate complex" refers to either
peptide-hair, peptide-skin, peptide-nail, or peptide teeth
complexes.
[0088] The term "MB.sub.50" refers to the concentration of the
binding peptide that gives a signal that is 50% of the maximum
signal obtained in an ELISA-based binding assay, as described in
Example 9. The MB.sub.50 provides an indication of the strength of
the binding interaction or affinity of the components of the
complex. The lower the value of MB.sub.50, the stronger the
interaction of the peptide with its corresponding substrate.
[0089] The term "binding affinity" refers to the strength of the
interaction of a binding peptide with its respective substrate. The
binding affinity is defined herein in terms of the MB.sub.50 value,
determined in an ELISA-based binding assay.
[0090] The term "nanoparticles" are herein defined as particles
with an average particle diameter of between 1 and 200 nm.
Preferably, the average particle diameter of the particles is
between about 1 and 40 nm. As used herein, "particle size" and
"particle diameter" have the same meaning. Nanoparticles include,
but are not limited to, metallic, semiconductor, polymer, or silica
particles.
[0091] The term "amino acid" refers to the basic chemical
structural unit of a protein or polypeptide. The following
abbreviations are used herein to identify specific amino acids:
TABLE-US-00001 Three-Letter One-Letter Amino Acid Abbreviation
Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic
acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E
Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine
Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser
S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0092] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences "Chimeric gene" refers to any
gene that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature. A
"foreign" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes.
[0093] "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments which are then
enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the
component nucleotides were assembled in vitro. Manual chemical
synthesis of DNA may be accomplished using well-established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codon bias of
the host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where
sequence information is available.
[0094] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Suitable regulatory sequences" refer
to nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, polyadenylation recognition sequences, RNA
processing sites, effector binding sites and stem-loop
structures.
[0095] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. Promoters
may be derived in their entirety from a native gene, or be composed
of different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental or physiological conditions. Promoters which cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". It is further recognized
that since in most cases the exact boundaries of regulatory
sequences have not been completely defined, DNA fragments of
different lengths may have identical promoter activity.
[0096] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0097] The term "transformation" refers to the transfer of a
nucleic acid fragment into the genome of a host organism, resulting
in genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0098] The term "host cell" refers to cell which has been
transformed or transfected, or is capable of transformation or
transfection by an exogenous polynucleotide sequence.
[0099] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA molecules. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitate
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0100] The term "phage" or "bacteriophage" refers to a virus that
infects bacteria. Altered forms may be used for the purpose of the
present invention. The preferred bacteriophage is derived from the
"wild" phage, called M13. The M13 system can grow inside a
bacterium, so that it does not destroy the cell it infects but
causes it to make new phages continuously. It is a single-stranded
DNA phage.
[0101] The term "phage display" refers to the display of functional
foreign peptides or small proteins on the surface of bacteriophage
or phagemid particles. Genetically engineered phage may be used to
present peptides as segments of their native surface proteins.
Peptide libraries may be produced by populations of phage with
different gene sequences.
[0102] "PCR" or "polymerase chain reaction" is a technique used for
the amplification of specific DNA segments (U.S. Pat. Nos.
4,683,195 and 4,800,159).
[0103] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis");
and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold
Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0104] The present invention provides diblock and triblock
peptide-based body surface coloring reagents which comprise at
least one body surface-binding peptide coupled to at least one
pigment-binding peptide, either directly or through a molecular
spacer. The body surface-binding peptide sequences and the
pigment-binding peptide sequences may be identified using
combinatorial methods, such as phage display. Additionally, the
body surface-binding peptide sequences may be empirically
generated. The diblock and triblock peptide-based reagents of the
invention may be prepared by covalently attaching the peptide
sequences, either directly or through a molecular spacer.
Alternatively, the entire diblock and triblock peptide-based
reagents may be produced biologically. The diblock and triblock
peptide-based body surface coloring reagents may be used in
conjunction with pigments to color body surfaces such as hair,
skin, nails, and teeth.
Body Surfaces
[0105] Body surfaces of the invention are any surface on the human
body that will serve as a substrate for a binding peptide. Typical
body surfaces include, but are not limited to hair, skin, nails,
teeth, gums, and the tissues of the oral cavity. In many cases the
body surfaces of the invention will be exposed to air, however in
some instances, the oral cavity for example, the surfaces will be
internal. Accordingly body surfaces may include layers of both
epithelial and well as endothelial cells.
[0106] Samples of body surfaces are available from a variety of
sources. For example, human hair samples are available
commercially, for example from International Hair Importers and
Products (Bellerose, N.Y.), in different colors, such as brown,
black, red, and blond, and in various types, such as
African-American, Caucasian, and Asian. Additionally, the hair
samples may be treated for example using hydrogen peroxide to
obtain bleached hair. Human skin samples may be obtained from
cadavers or in vitro human skin cultures. Additionally, pig skin,
available from butcher shops and supermarkets, Vitro-Skin.RTM.,
available from IMS Inc. (Milford, Conn.), and EpiDerm.TM.,
available from MatTek Corp. (Ashland, Mass.), are good substitutes
for human skin. Human fingernails and toenails may be obtained from
volunteers. Extracted human teeth and false teeth may be obtained
from Dental offices. Additionally, hydroxyapatite, available in
many forms for example from Berkeley Advanced Biomaterials, Inc.
(San Leandro, Calif.), may be used as a model for human teeth.
Body Surface-Binding Peptides
[0107] Body surface-binding peptides as defined herein are peptide
sequences that specifically bind with high affinity to specific
body surfaces, including, but not limited to hair, nails, teeth,
gums, skin, and the tissues of the oral cavity, for example.
Suitable body surface-binding peptide sequences may be selected
using combinatorial methods that are well known in the art or may
be empirically generated. The body surface binding peptides of the
invention have a binding affinity for their respective substrate,
as measured by MB.sub.50 values, of less than or equal to about
10.sup.-2M, less than or equal to about 10.sup.-3 M, less than or
equal to about 10.sup.-4 M, less than or equal to about 10.sup.-5
M, preferably less than or equal to about 10.sup.-6 M, and more
preferably less than or equal to about 10.sup.-7 M.
[0108] Combinatorially generated body surface-binding peptides of
the present invention are from about 7 amino acids to about 50
amino acids, more preferably, from about 7 amino acids to about 25
amino acids in length. The body surface-binding peptides of the
present invention may be generated randomly and then selected
against a specific body surface, for example, hair, skin, nail, or
tooth sample, based upon their binding affinity for the surface of
interest. The generation of random libraries of peptides is well
known and may be accomplished by a variety of techniques including,
bacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA
78(7):4520-4524 (1981), and Helfman et al., Proc. Natl. Acad. Sci.
USA 80(1):31-35, (1983)), yeast display (Chien et al., Proc Natl
Acad Sci USA 88(21):9578-82 (1991)), combinatorial solid phase
peptide synthesis (U.S. Pat. No. 5,449,754, U.S. Pat. No.
5,480,971, U.S. Pat. No. 5,585,275, U.S. Pat. No. 5,639,603), and
phage display technology (U.S. Pat. No. 5,223,409, U.S. Pat. No.
5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500).
Techniques to generate such biological peptide libraries are
described in Dani, M., J. of Receptor & Signal Transduction
Res., 21(4):447-468 (2001). Additionally, phage display libraries
are available commercially from companies such as New England
BioLabs (Beverly, Mass.).
[0109] A preferred method to randomly generate peptides is by phage
display. Phage display is an in vitro selection technique in which
a peptide or protein is genetically fused to a coat protein of a
bacteriophage, resulting in display of fused peptide on the
exterior of the phage virion, while the DNA encoding the fusion
resides within the virion. This physical linkage between the
displayed peptide and the DNA encoding it allows screening of vast
numbers of variants of peptides, each linked to a corresponding DNA
sequence, by a simple in vitro selection procedure called
"biopanning". In its simplest form, biopanning is carried out by
incubating the pool of phage-displayed variants with a target of
interest that has been immobilized on a plate or bead, washing away
unbound phage, and eluting specifically bound phage by disrupting
the binding interactions between the phage and the target. The
eluted phage is then amplified in vivo and the process is repeated,
resulting in a stepwise enrichment of the phage pool in favor of
the tightest binding sequences. After 3 or more rounds of
selection/amplification, individual clones are characterized by DNA
sequencing.
[0110] More specifically, after a suitable library of peptides has
been generated or purchased, the library is then contacted with an
appropriate amount of the test substrate, specifically a body
surface sample. The library of peptides is dissolved in a suitable
solution for contacting the sample. The body surface sample may be
suspended in the solution or may be immobilized on a plate or bead.
A preferred solution is a buffered aqueous saline solution
containing a surfactant. A suitable solution is Tris-buffered
saline (TBS) with 0.5% Tween.RTM. 20. The solution may additionally
be agitated by any means in order to increase the mass transfer
rate of the peptides to body surface sample, thereby shortening the
time required to attain maximum binding.
[0111] Upon contact, a number of the randomly generated peptides
will bind to the body surface sample to form a peptide-body-surface
complex, for example a peptide-hair, peptide-skin, peptide-nail, or
peptide-tooth complex. Unbound peptide may be removed by washing.
After all unbound material is removed, peptides having varying
degrees of binding affinities for the test surface may be
fractionated by selected washings in buffers having varying
stringencies. Increasing the stringency of the buffer used
increases the required strength of the bond between the peptide and
body surface in the peptide-body surface complex.
[0112] A number of substances may be used to vary the stringency of
the buffer solution in peptide selection including, but not limited
to, acidic pH (1.5-3.0); basic pH (10-12.5); high salt
concentrations such as MgCl.sub.2 (3-5 M) and LiCl (5-10 M); water;
ethylene glycol (25-50%); dioxane (5-20%); thiocyanate (1-5 M);
guanidine (2-5 M); urea (2-8 M); and various concentrations of
different surfactants such as SDS (sodium dodecyl sulfate), DOC
(sodium deoxycholate), Nonidet P-40, Triton X-100, Tween.RTM. 20,
wherein Tween.RTM. 20 is preferred. These substances may be
prepared in buffer solutions including, but not limited to,
Tris-HCl, Tris-buffered saline, Tris-borate, Tris-acetic acid,
triethylamine, phosphate buffer, and glycine-HCl, wherein
Tris-buffered saline solution is preferred.
[0113] It will be appreciated that peptides having increasing
binding affinities for body surface substrates may be eluted by
repeating the selection process using buffers with increasing
stringencies. The eluted peptides can be identified and sequenced
by any means known in the art.
[0114] Thus, the following method for generating the body
surface-binding peptides, for example, hair-binding peptides,
skin-binding peptides, nail-binding peptides, or tooth-binding
peptides, may be used. A library of combinatorially generated
phage-peptides is contacted with the body surface of interest, to
form phage peptide-body surface complexes. The
phage-peptide-body-surface complex is separated from uncomplexed
peptides and unbound substrate, and the bound phage-peptides from
the phage-peptide-body surface complexes is eluted from the
complex, preferably by acid treatment. Then, the eluted
phage-peptides are identified and sequenced. To identify peptide
sequences that bind to one substrate but not to another, for
example peptides that bind to hair, but not to skin or peptides
that bind to skin, but not to hair, a subtractive panning step is
added. Specifically, the library of combinatorially generated
phage-peptides is first contacted with the non-target to remove
phage-peptides that bind to it. Then, the non-binding
phage-peptides are contacted with the desired substrate and the
above process is followed. Alternatively, the library of
combinatorially generated phage-peptides may be contacted with the
non-target and the desired substrate simultaneously. Then, the
phage-peptide-body surface complexes are separated from the
phage-peptide-non-target complexes and the method described above
is followed for the desired phage-peptide-body surface
complexes.
[0115] In one embodiment, a modified phage display screening method
for isolating peptides with a higher affinity for body surfaces is
used. In the modified method, the phage-peptide-body surface
complexes are formed as described above. Then, these complexes are
treated with an elution buffer. Any of the elution buffers
described above may be used. Preferably, the elution buffer is an
acidic solution. Then, the remaining, elution-resistant
phage-peptide-body surface complexes are used to directly infect a
bacterial host cell, such as E. coli ER2738. The infected host
cells are grown in an appropriate growth medium, such as LB
(Luria-Bertani) medium, and this culture is spread onto agar,
containing a suitable growth medium, such as LB medium with IPTG
(isopropyl .beta.-D-thiogalactopyranoside) and S-Gal.TM.. After
growth, the plaques are picked for DNA isolation and are sequenced
to identify the peptide sequences with a high binding affinity for
the body surface of interest.
[0116] In another embodiment, PCR may be used to identify the
elution-resistant phage-peptides from the modified phage display
screening method, described above, by directly carrying out PCR on
the phage-peptide-body surface complexes using the appropriate
primers, as described by Janssen et al. in U.S. Patent Application
Publication No. 2003/0152976, which is incorporated herein by
reference.
[0117] Hair-binding, skin-binding, and nail-binding peptides have
been identified using the above methods, as described by Huang et
al. in copending and commonly owned U.S. Patent Application
Publication No. 2005/0050656, and U.S. Patent Application
Publication No. 2005/0226839, both of which are incorporated herein
by reference. Specifically, binding peptides were isolated that
have a high affinity for normal brown hair, given as SEQ ID
NOs:3-18, 28-38, 40-56, and 64; shampoo resistant peptides having
affinity for normal brown hair, given as SEQ ID NO:66, 69 and 70;
bleached hair, given as SEQ ID NOs:7, 8, 19-27, 38-40, 43, 44, 47,
57, 58, and 59, fingernail, given as SEQ ID NOs:53 and 60; and
skin, given as SEQ ID NO:61. Additionally, the fingernail-binding
peptides were found to bind to bleached hair and may be used in the
peptide-based hair reagents of the invention. The bleached
hair-binding peptides will bind to fingernails and may be used in
the peptide-based nail reagents of the invention.
[0118] Alternatively, hair and skin-binding peptide sequences may
be generated empirically by designing peptides that comprise
positively charged amino acids, which can bind to hair and skin via
electrostatic interaction, as described by Rothe et al. (WO
2004/000257). The empirically generated hair and skin-binding
peptides have between about 4 amino acids to about 50 amino acids,
preferably from about 4 to about 25 amino acids, and comprise at
least about 40 mole % positively charged amino acids, such as
lysine, arginine, and histidine. Peptide sequences containing
tripeptide motifs such as HRK, RHK, HKR, RKH, KRH, KHR, HKX, KRX,
RKX, HRX, KHX and RHX are most preferred where X can be any natural
amino acid but is most preferably selected from neutral side chain
amino acids such as glycine, alanine, proline, leucine, isoleucine,
valine and phenylalanine. In addition, it should be understood that
the peptide sequences must meet other functional requirements in
the end use including solubility, viscosity and compatibility with
other components in a formulated product and will therefore vary
according to the needs of the application. In some cases the
peptide may contain up to 60 mole % of amino acids not comprising
histidine, lysine or arginine. Suitable empirically generated
hair-binding and skin peptides include, but are not limited to, SEQ
ID NOs:105-109.
Pigment-Binding Peptides
[0119] Pigment-binding peptides (PBP) as defined herein are peptide
sequences that specifically bind with high affinity to pigments.
The pigment-binding peptides are from about 5 amino acids to 50
amino acids, more preferably, from about 7 amino acids to about 12
amino acids in length.
[0120] Suitable pigment-binding peptide sequences may be selected
using methods that are well known in the art. For example,
pigment-binding peptides may be generated randomly and then
selected against a specific pigment based upon their binding
affinity for the pigment of interest, as described by O'Brien et
al. in U.S. Patent Application Publication No. 2005/0054752,
incorporated herein by reference. That method is similar to that
described above for the selection of body surface-binding
peptides.
[0121] As used herein, the term "pigment" means an insoluble
colorant. A wide variety of organic and inorganic pigments alone or
in combination may be used in the present invention. Examples of
suitable pigments include, but are not limited to D&C Red No.
36, D&C Red No. 30, D&C Orange No. 17, Green 3 Lake, Ext.
Yellow 7 Lake, Orange 4 Lake, and Red 28 Lake; the calcium lakes of
D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&C Red
No. 12, the strontium lake D&C Red No. 13, the aluminum lakes
of FD&C Yellow No. 5, of FD&C Yellow No. 6, of FD&C No.
40, of D&C Red Nos. 21, 22, 27, and 28, of FD&C Blue No. 1,
of D&C Orange No. 5, of D&C Yellow No. 10, the zirconium
lake of D&C Red No. 33; Cromophthal.RTM. Yellow 131AK (Ciba
Specialty Chemicals), Sunfast.RTM. Magenta 122 (Sun Chemical) and
Sunfast.RTM. Blue 15:3 (Sun Chemical), iron oxides, calcium
carbonate, aluminum hydroxide, calcium sulfate, kaolin, ferric
ammonium ferrocyamide, magnesium carbonate, carmine, barium
sulfate, mica, bismuth oxychloride, zinc stearate, manganese
violet, chromium oxide, titanium dioxide, titanium dioxide
nanoparticles, zinc oxide, barium oxide, ultramarine blue, bismuth
citrate, and white minerals such as hydroxyapatite, and Zircon
(zirconium silicate), and carbon black particles.
[0122] Examples of suitable pigment-binding peptides include, but
are not limited to, those described by O'Brien et al., supra, that
have a high affinity for the pigments carbon black, given as SEQ ID
NOs:110-113, Cromophthal.RTM. Yellow, given as SEQ ID NOs:114-122,
Sunfast.RTM. Magenta, given as SEQ ID NOs:123-125, and Sunfast.RTM.
Blue, given as SEQ ID NOs:122, 126-134, and those described by
Nomoto et al. in EP1275728 that have a high affinity for carbon
black, copper phthalocyanine, titanium dioxide, and silicon
dioxide.
Production of Binding Peptides
[0123] The body surface-binding peptides and pigment-binding
peptides of the present invention may be prepared using standard
peptide synthesis methods, which are well known in the art (see for
example Stewart et al., Solid Phase Peptide Synthesis, Pierce
Chemical Co., Rockford, Ill., 1984; Bodanszky, Principles of
Peptide Synthesis, Springer-Verlag, New York, 1984; and Pennington
et al., Peptide Synthesis Protocols, Humana Press, Totowa, N.J.,
1994). Additionally, many companies offer custom peptide synthesis
services.
[0124] Alternatively, the peptides of the present invention may be
prepared using recombinant DNA and molecular cloning techniques.
Genes encoding the hair-binding, skin-binding or nail-binding
peptides may be produced in heterologous host cells, particularly
in the cells of microbial hosts.
[0125] Preferred heterologous host cells for expression of the
binding peptides of the present invention are microbial hosts that
can be found broadly within the fungal or bacterial families and
which grow over a wide range of temperature, pH values, and solvent
tolerances. Because transcription, translation, and the protein
biosynthetic apparatus are the same irrespective of the cellular
feedstock, functional genes are expressed irrespective of carbon
feedstock used to generate cellular biomass. Examples of host
strains include, but are not limited to, fungal or yeast species
such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida,
Hansenula, or bacterial species such as Salmonella, Bacillus,
Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas,
Methylomonas, Methylobacter, Alcaligenes, Synechocystis, Anabaena,
Thiobacillus, Methanobacterium and Klebsiella.
[0126] A variety of expression systems can be used to produce the
peptides of the present invention. Such vectors include, but are
not limited to, chromosomal, episomal and virus-derived vectors,
e.g., vectors derived from bacterial plasmids, from bacteriophage,
from transposons, from insertion elements, from yeast episoms, from
viruses such as baculoviruses, retroviruses and vectors derived
from combinations thereof such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagemids. The
expression system constructs may contain regulatory regions that
regulate as well as engender expression. In general, any system or
vector suitable to maintain, propagate or express polynucleotide or
polypeptide in a host cell may be used for expression in this
regard. Microbial expression systems and expression vectors contain
regulatory sequences that direct high level expression of foreign
proteins relative to the growth of the host cell. Regulatory
sequences are well known to those skilled in the art and examples
include, but are not limited to, those which cause the expression
of a gene to be turned on or off in response to a chemical or
physical stimulus, including the presence of regulatory elements in
the vector, for example, enhancer sequences. Any of these could be
used to construct chimeric genes for production of the any of the
binding peptides of the present invention. These chimeric genes
could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the
peptides.
[0127] Vectors or cassettes useful for the transformation of
suitable host cells are well known in the art. Typically the vector
or cassette contains sequences directing transcription and
translation of the relevant gene, one or more selectable markers,
and sequences allowing autonomous replication or chromosomal
integration. Suitable vectors comprise a region 5' of the gene,
which harbors transcriptional initiation controls and a region 3'
of the DNA fragment which controls transcriptional termination. It
is most preferred when both control regions are derived from genes
homologous to the transformed host cell, although it is to be
understood that such control regions need not be derived from the
genes native to the specific species chosen as a production host.
Selectable marker genes provide a phenotypic trait for selection of
the transformed host cells such as tetracycline or ampicillin
resistance in E. coli.
[0128] Initiation control regions or promoters which are useful to
drive expression of the chimeric gene in the desired host cell are
numerous and familiar to those skilled in the art. Virtually any
promoter capable of driving the gene is suitable for producing the
binding peptides of the present invention including, but not
limited to: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1,
TRP1, URA3, LEU2, ENO, TPI (useful for expression in
Saccharomyces); AOX1 (useful for expression in Pichia); and lac,
ara, tet, trp, IP.sub.L, IP.sub.R, T7, tac, and trc (useful for
expression in Escherichia coli) as well as the amy, apr, npr
promoters and various phage promoters useful for expression in
Bacillus.
[0129] Termination control regions may also be derived from various
genes native to the preferred hosts. Optionally, a termination site
may be unnecessary, however, it is most preferred if included.
[0130] The vector containing the appropriate DNA sequence as
described supra, as well as an appropriate promoter or control
sequence, may be employed to transform an appropriate host to
permit the host to express the peptide of the present invention.
Cell-free translation systems can also be employed to produce such
peptides using RNAs derived from the DNA constructs of the present
invention. Optionally it may be desired to produce the instant gene
product as a secretion product of the transformed host. Secretion
of desired proteins into the growth media has the advantages of
simplified and less costly purification procedures. It is well
known in the art that secretion signal sequences are often useful
in facilitating the active transport of expressible proteins across
cell membranes. The creation of a transformed host capable of
secretion may be accomplished by the incorporation of a DNA
sequence that codes for a secretion signal which is functional in
the production host. Methods for choosing appropriate signal
sequences are well known in the art (see for example EP 546049 and
WO 9324631). The secretion signal DNA or facilitator may be located
between the expression-controlling DNA and the instant gene or gene
fragment, and in the same reading frame with the latter.
Diblock and Triblock Peptide-Based Body Surface Coloring
Reagents
[0131] The peptide-based diblock and triblock peptide-based body
surface coloring reagents of the present invention are formed by
coupling at least one body surface-binding peptide to at least one
pigment-binding peptide, either directly or through a molecular
spacer. The body surface-binding peptide part of the reagent binds
strongly to the body surface, while the pigment-binding sequence
binds strongly to the pigment, thereby attaching the pigment to the
body surface. The diblock and triblock peptide-based body surface
coloring reagents of the invention are from about 14 to about 200
amino acids in length, preferably about 30 to about 130 amino acids
in length.
[0132] Suitable body surface-binding peptides are described above
and include, but are not limited to hair-binding, skin-binding,
nail-binding, and tooth-binding peptides selected by the screening
methods described above, and empirically generated hair and
skin-binding peptides, as described above. Additionally, any known
body surface binding peptide may be used, including hair-binding
peptides such as SEQ ID NO:1, and skin-binding peptides such as SEQ
ID NO:2, described by Janssen et al. in U.S. Patent Application
Publication No. 2003/0152976, and hair-binding peptides such as SEQ
ID NOs:76-98, and skin-binding peptides such as SEQ ID NOs:99-104,
described by Janssen et al. in WO 04048399, both of which are
incorporated herein by reference. Additionally, hair conditioner
resistant hair-binding peptides such as SEQ ID NO:75, described by
Wang et al. (U.S. Patent Application No. 60/657,496), and hair
conditioner and shampoo resistant hair-binding peptides such as SEQ
ID NOs:153-156, as described by O'Brien et al. (U.S. patent
application Ser. No. 11/251,715), may be used.
[0133] Suitable pigment-binding peptides are described above and
include pigment-binding peptides selected by the screening methods
described above. Additionally, any known pigment-binding peptide
may be used, such as the peptides that bind to carbon black, copper
phthalocyanine, titanium dioxide, and silicon dioxide, described by
Nomoto et al. in EP1275728.
[0134] The diblock and triblock peptide-based body surface coloring
reagents of the present invention are prepared by coupling at least
one body surface-binding peptide to at least one pigment-binding
peptide, either directly or via an optional spacer. The coupling
interaction may be a covalent bond or a non-covalent interaction,
such as hydrogen bonding, electrostatic interaction, hydrophobic
interaction, or Van der Waals interaction. In the case of a
non-covalent interaction, the peptide-based body surface coloring
reagents may be prepared by mixing at least one body
surface-binding peptide, at least one pigment-binding peptide and
the optional spacer (if used) and allowing sufficient time for the
interaction to occur. The unbound materials may be separated from
the resulting peptide-based body surface coloring reagent using
methods known in the art, for example, gel permeation
chromatography.
[0135] The peptide-based body surface coloring reagents of the
invention may also be prepared by covalently attaching at least one
body surface-binding peptide to at least one pigment-binding
peptide, either directly or through a spacer. Any known peptide or
protein conjugation chemistry may be used to form the peptide-based
body surface coloring reagents of the invention. Conjugation
chemistries are well-known in the art (see for example, Hermanson,
Bioconjugate Techniques, Academic Press, New York (1996)). Suitable
coupling agents include, but are not limited to, carbodiimide
coupling agents, diacid chlorides, diisocyanates and other
difunctional coupling reagents that are reactive toward terminal
amine and/or carboxylic acid groups on the peptides. The preferred
coupling agents are carbodiimide coupling agents, such as
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and
N,N'-dicyclohexyl-carbodiimide (DCC), which may be used to activate
carboxylic acid groups. Additionally, it may be necessary to
protect reactive amine or carboxylic acid groups on the peptides to
produce the desired structure for the peptide-based body surface
coloring reagent. The use of protecting groups for amino acids,
such as t-butyloxycarbonyl (t-Boc), are well known in the art (see
for example Stewart et al., supra; Bodanszky, supra; and Pennington
et al., supra).
[0136] Additionally, diblock peptide-based body surface coloring
reagents consisting of at least one body surface binding peptide
and at least one pigment-binding peptide may be prepared using the
recombinant DNA and molecular cloning techniques described
supra.
[0137] It may also be desirable to couple the body surface-binding
peptide to the pigment-binding peptide via a spacer to form a
triblock body surface coloring reagent. The spacer serves to
separate the binding peptide sequences to ensure that the binding
affinity of the individual peptides is not adversely affected by
the coupling. The spacer may also provide other desirable
properties such as hydrophilicity, hydrophobicity, or a means for
cleaving the peptide sequences to facilitate removal of the
coloring agent.
[0138] The spacer may be any of a variety of molecules, such as
alkyl chains, phenyl compounds, ethylene glycol, amides, esters and
the like. Preferred spacers are hydrophilic and have a chain length
from 1 to about 100 atoms, more preferably, from 2 to about 30
atoms. Examples of preferred spacers include, but are not limited
to ethanol amine, ethylene glycol, polyethylene with a chain length
of 6 carbon atoms, polyethylene glycol with 3 to 6 repeating units,
phenoxyethanol, propanolamide, butylene glycol,
butyleneglycolamide, propyl phenyl chains, and ethyl, propyl,
hexyl, steryl, cetyl, and palmitoyl alkyl chains. The spacer may be
covalently attached to the body surface-binding and pigment-binding
peptide sequences using any of the coupling chemistries described
above. In order to facilitate incorporation of the spacer, a
bifunctional cross-linking agent that contains a spacer and
reactive groups at both ends for coupling to the peptides may be
used. Suitable bifunctional cross-linking agents are well known in
the art and include, but are not limited to diamines, such a as
1,6-diaminohexane; dialdehydes, such as glutaraldehyde; bis
N-hydroxysuccinimide esters, such as ethylene glycol-bis(succinic
acid N-hydroxysuccinimide ester), disuccinimidyl glutarate,
disuccinimidyl suberate, and ethylene
glycol-bis(succinimidylsuccinate); diisocyanates, such as
hexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyl
diglycidyl ether; dicarboxylic acids, such as succinyldisalicylate;
and the like. Heterobifunctional cross-linking agents, which
contain a different reactive group at each end, may also be used.
Examples of heterobifunctional cross-linking agents include, but
are not limited to compounds having the following structure:
##STR1## where: R.sub.1 is H or a substituent group such as
--SO.sub.3Na, --NO.sub.2, or --Br; and R.sub.2 is a spacer such as
--CH.sub.2CH.sub.2 (ethyl), --(CH.sub.2).sub.3 (propyl), or
--(CH.sub.2).sub.3C.sub.6H.sub.5 (propyl phenyl). An example of
such a heterobifunctional cross-linking agent is
3-maleimidopropionic acid N-hydroxysuccinimide ester. The
N-hydroxysuccinimide ester group of these reagents reacts with
amine groups on one peptide, while the maleimide group reacts with
thiol groups present on the other peptide. A thiol group may be
incorporated into the peptide by adding at least one cysteine group
to at least one end of the binding peptide sequence (i.e., the
C-terminal end or N-terminal end). Several spacer amino acid
residues, such as glycine, may be incorporated between the binding
peptide sequence and the terminal cysteine to separate the reacting
thiol group from the binding sequence. Moreover, at least one
lysine residue may be added to at least one end of the binding
peptide sequence, i.e., the C-terminal end or the N-terminal end,
to provide an amine group for coupling.
[0139] Additionally, the spacer may be a peptide comprising any
amino acid and mixtures thereof. The preferred peptide spacers
comprise the amino acids glycine, alanine, and serine, and mixtures
thereof. In addition, the peptide spacer may contain a specific
enzyme cleavage site, such as the protease Caspase 3 site, given by
SEQ ID NO:65, which allows for the enzymatic removal of the pigment
from the hair. The peptide spacer may be from 1 to about 50 amino
acids, preferably from 1 to about 20 amino acids in length.
Examples of suitable spacers include, but are not limited to, the
sequences given by SEQ ID NOs:135-137. These peptide spacers may be
linked to the binding peptide sequences by any method known in the
art. For example, the entire triblock peptide-based body surface
coloring reagent may be prepared using the standard peptide
synthesis methods described supra. In addition, the binding
peptides and peptide spacer block may be combined using
carbodiimide coupling agents (see for example, Hermanson,
Bioconjugate Techniques, Academic Press, New York (1996)), diacid
chlorides, diisocyanates and other difunctional coupling reagents
that are reactive to terminal amine and/or carboxylic acid groups
on the peptides, as described above. Alternatively, the entire
triblock peptide-based body surface coloring reagent may be
prepared using the recombinant DNA and molecular cloning techniques
described supra. The spacer may also be a combination of a peptide
spacer and an organic spacer molecule, which may be prepared using
the methods described above. Examples of triblock body surface
coloring reagents include, but are not limited to, the sequences
given as SEQ ID NOs:138-147.
[0140] It may also be desirable to have multiple copies of the body
surface-binding peptide and the pigment-binding peptide coupled
together to enhance the interaction between the peptide-based body
surface coloring reagent and the body surface and the pigment, as
described by Huang et al. (U.S. Patent Application Publication No.
2005/0050656). Either multiple copies of the same body
surface-binding peptide and pigment-binding peptide or a
combination of different body surface-binding peptides and
pigment-binding peptides may be used. The multi-copy peptide-based
body surface coloring reagents may comprise various spacers as
described above. Examples of multi-copy body surface-binding
peptide-pigment-binding peptide body surface coloring reagents
include, but are not limited to, the sequences given as SEQ ID
NOs:144, 145, and 147.
[0141] In one embodiment of the invention, the peptide-based body
surface coloring reagent is a diblock composition comprising a body
surface-binding peptide (BSBP) and a pigment-binding peptide (PBP),
having the general structure [(BSBP).sub.m-(PBP).sub.n].sub.x,
where n and m independently range from 1 to about 10, preferably
from 1 to about 5, and x may be 1 to about 10.
[0142] In another embodiment, the peptide-based body surface
coloring reagent comprises a molecular spacer (S) separating the
body surface-binding peptide from the pigment-binding peptide, as
described above. Multiple copies of the body surface-binding
peptide and the pigment-binding peptide may also be used and the
multiple copies of the body surface-binding peptide and the
pigment-binding peptide may be separated from themselves and from
each other by molecular spacers. In this embodiment, the
peptide-based body surface coloring reagent is a triblock
composition comprising a body surface-binding peptide, a spacer,
and pigment-binding peptide, having the general structure
[[(BSBP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z].sub.y,
where n, m, x, and z independently range from 1 to about 10, y is
from 1 to about 5, and where q and r are each independently 0 or 1,
provided that both q and r are not 0. Preferably, m and n
independently range from 1 to about 5, and x and z range from 1 to
about 3.
[0143] In another embodiment, the body surface-binding peptide is a
hair-binding peptide and the peptide-based body surface coloring
reagent is a diblock composition comprising the hair-binding
peptide (HBP) and a pigment-binding peptide (PBP), having the
general structure [(HBP).sub.m-(PBP).sub.n].sub.x where n and m
independently range from 1 to about 10, preferably from 1 to about
5, and x may be 1 to about 10.
[0144] In another embodiment, the body surface-binding peptide is a
hair-binding peptide and the peptide-based body surface coloring
reagent is a triblock composition comprising the hair-binding
peptide (HBP), a spacer (S), and a pigment-binding peptide (PBP),
having the general structure [[(H
BP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z]y, where n, m,
x, and z independently range from 1 to about 10, y is from 1 to
about 5, and where q and r are each independently 0 or 1, provided
that both q and r are not 0. Preferably, m and n independently
range from 1 to about 5, and x and z range from 1 to about 3.
[0145] In another embodiment, the body surface-binding peptide is a
skin-binding peptide and the peptide-based body surface coloring
reagent is a diblock composition comprising the skin-binding
peptide (SBP) and a pigment-binding peptide (PBP), having the
general structure [(SBP).sub.m-(PBP).sub.n].sub.x, where n and m
independently range from 1 to about 10, preferably from 1 to about
5, and x may be 1 to about 10.
[0146] In another embodiment, the body surface-binding peptide is a
skin-binding peptide and the peptide-based body surface coloring
reagent is a triblock composition comprising the skin-binding
peptide (SBP), a spacer (S), and a pigment-binding peptide (PBP),
having the general structure
[[(SBP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z]y, where
n, m, x, and z independently range from 1 to about 10, y is from 1
to about 5, and where q and r are each independently 0 or 1,
provided that both q and r are not 0. Preferably, m and n
independently range from 1 to about 5, and x and z range from 1 to
about 3.
[0147] In another embodiment, the body surface-binding peptide is a
nail-binding peptide and the peptide-based body surface coloring
reagent is a diblock composition comprising the nail-binding
peptide (NBP) and a pigment-binding peptide (PBP), having the
general structure [(NBP).sub.m-(PBP).sub.n].sub.x where n and m
independently range from 1 to about 10, preferably from 1 to about
5, and x may be 1 to about 10.
[0148] In another embodiment, the body surface-binding peptide is a
nail-binding peptide and the peptide-based body surface coloring
reagent is a triblock composition comprising the nail-binding
peptide (NBP), a spacer (S), and a pigment-binding peptide (PBP),
having the general structure [[(N
BP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z]y, where n, m,
x, and z independently range from 1 to about 10, y is from 1 to
about 5, and where q and r are each independently 0 or 1, provided
that both q and r are not 0. Preferably, m and n independently
range from 1 to about 5, and x and z range from 1 to about 3.
[0149] In another embodiment, the body surface-binding peptide is a
tooth-binding peptide and the peptide-based body surface coloring
reagent is a diblock composition comprising the tooth-binding
peptide (TBP) and a pigment-binding peptide (PBP), having the
general structure [(TBP).sub.m-(PBP).sub.n].sub.x where n and m
independently range from 1 to about 10, preferably from 1 to about
5, and x may be 1 to about 10.
[0150] In another embodiment, the body surface-binding peptide is a
tooth-binding peptide and the peptide-based body surface coloring
reagent is a triblock composition comprising the tooth-binding
peptide (TBP), a spacer (S), and a pigment-binding peptide (PBP),
having the general structure
[[(TBP).sub.m-S.sub.q].sub.x-[(PBP).sub.n-S.sub.r].sub.z]y, where
n, m, x, and z independently range from 1 to about 10, y is from 1
to about 5, and where q and r are each independently 0 or 1,
provided that both q and r are not 0. Preferably, m and n
independently range from 1 to about 5, and x and z range from 1 to
about 3.
[0151] It should be understood that as used herein, BSBP, HBP, SBP,
NBP, TBP, and PBP are generic designations and are not meant to
refer to a single body surface-binding peptide, hair-binding
peptide, skin-binding peptide, nail-binding peptide, tooth-binding
or pigment-binding peptide sequence, respectively. Where m or n as
used above, is greater than 1, it is well within the scope of the
invention to provide for the situation where a series of body
surface-binding peptides of different sequences and pigment-binding
peptides of different sequences may form a part of the composition.
Additionally, S is a generic term and is not meant to refer to a
single spacer. Where x and y, as used above for the triblock
compositions, are greater than 1, it is well within the scope of
the invention to provide for the situation where a series of
different spacers may form a part of the composition. It should
also be understood that these structures do not necessarily
represent a covalent bond between the peptides and the optional
molecular spacer. As described above, the coupling interaction
between the peptides and the optional spacer may be either covalent
or non-covalent.
Personal Care Compositions
[0152] The diblock and triblock peptide-based body surface coloring
reagents of the invention may be used in personal care compositions
in conjunction with one or more pigments to color body surfaces,
such as hair, skin, nails, and teeth. The body surface-binding
peptide block of the peptide-based body surface coloring agent has
an affinity for the body surface, while the pigment-binding peptide
block has an affinity for the pigment used, thereby attaching the
pigment to the body surface. The peptide-based body surface
coloring reagent may be present in the same composition as the
pigment, or the peptide-based body surface coloring reagent and the
pigment may be present in two different personal care compositions
that are applied to the body surface in any order, as described
below. Personal care compositions include, but are not limited to,
hair care compositions, hair coloring compositions, skin care
compositions, cosmetic compositions, nail polish compositions, and
oral care compositions.
[0153] Hair Care Compositions
[0154] In one embodiment, the peptide-based body surface coloring
reagent is a component of a hair care composition and the
peptide-based body surface coloring reagent comprises at least one
hair-binding peptide. Hair care compositions are herein defined as
compositions for the treatment of hair including, but not limited
to, shampoos, conditioners, rinses, lotions, aerosols, gels, and
mousses. An effective amount of the peptide-based body surface
coloring reagent for use in hair care compositions is a
concentration of about 0.01% to about 10%, preferably about 0.01%
to about 5% by weight relative to the total weight of the
composition. This proportion may vary as a function of the type of
hair care composition. Additionally, the hair care composition may
further comprise at least one pigment. Suitable pigments are
described above. The concentration of the peptide-based body
surface coloring reagent in relation to the concentration of the
pigment may need to be optimized for best results. Additionally, a
mixture of different peptide-based body surface coloring reagents
having an affinity for different pigments may be used in the
composition. The peptide-based body surface coloring reagents in
the mixture need to be chosen so that there is no interaction
between the peptides that mitigates the beneficial effect. Suitable
mixtures of peptide-based body surface coloring reagents may be
determined by one skilled in the art using routine experimentation.
If a mixture of peptide-based body surface coloring reagents is
used in the composition, the total concentration of the reagents is
about 0.01% to about 10% by weight relative to the total weight of
the composition.
[0155] The composition may further comprise a cosmetically
acceptable medium for hair care compositions, examples of which are
described by Philippe et al. in U.S. Pat. No. 6,280,747, and by
Omura et al. in U.S. Pat. No. 6,139,851 and Cannell et al. in U.S.
Pat. No. 6,013,250, all of which are incorporated herein by
reference. For example, these hair care compositions can be
aqueous, alcoholic or aqueous-alcoholic solutions, the alcohol
preferably being ethanol or isopropanol, in a proportion of from
about 1 to about 75% by weight relative to the total weight for the
aqueous-alcoholic solutions. Additionally, the hair care
compositions may contain one or more conventional cosmetic or
dermatological additives or adjuvants including, but not limited
to, antioxidants, preserving agents, fillers, surfactants, UVA
and/or UVB sunscreens, fragrances, thickeners, wetting agents and
anionic, nonionic or amphoteric polymers, and dyes.
[0156] Hair Coloring Compositions
[0157] In another embodiment, the peptide-based body surface
coloring reagent is a component of a hair coloring composition and
the peptide-based body surface coloring reagent comprises at least
one hair binding peptide. Hair coloring compositions are herein
defined as compositions for the coloring or dyeing of hair, which
comprise one or more coloring agents. Coloring agents as herein
defined are any dye, pigment, and the like that may be used to
change the color of a body surface, such as hair, skin, nails, or
teeth. Hair coloring agents are well known in the art (see for
example Green et al. supra, CFTA International Color Handbook,
2.sup.nd ed., Micelle Press, England (1992) and Cosmetic Handbook,
US Food and Drug Administration, FDA/IAS Booklet (1992)), and are
available commercially from various sources (for example Bayer,
Pittsburgh, Pa.; Ciba-Geigy, Tarrytown, N.Y.; ICI, Bridgewater,
N.J.; Sandoz, Vienna, Austria; BASF, Mount Olive, N.J.; and
Hoechst, Frankfurt, Germany).
[0158] An effective amount of a peptide-based body surface coloring
reagent for use in a hair coloring composition is herein defined as
a proportion of from about 0.01% to about 20% by weight relative to
the total weight of the composition. Additionally, a mixture of
different peptide-based body surface coloring reagents having an
affinity for different pigments may be used in the composition. The
peptide-based body surface coloring reagents in the mixture need to
be chosen so that there is no interaction between the peptides that
mitigates the beneficial effect. Suitable mixtures of peptide-based
body surface coloring reagents may be determined by one skilled in
the art using routine experimentation. If a mixture of
peptide-based body surface coloring reagents is used in the
composition, the total concentration of the reagents is about 0.01%
to about 20% by weight relative to the total weight of the
composition.
[0159] Components of a cosmetically acceptable medium for hair
coloring compositions are described by Dias et al., in U.S. Pat.
No. 6,398,821 and by Deutz et al., in U.S. Pat. No. 6,129,770, both
of which are incorporated herein by reference. For example, hair
coloring compositions may contain sequestrants, stabilizers,
thickeners, buffers, carriers, surfactants, solvents, antioxidants,
polymers, and conditioners.
[0160] Skin Care Compositions
[0161] In another embodiment, the peptide-based body surface
coloring reagent is a component of a skin care composition and the
peptide-based body surface coloring reagent comprises at least one
skin-binding peptide. Skin care compositions are herein defined as
compositions for the treatment of skin including, but not limited
to, skin care, skin cleansing, make-up, and anti-wrinkle products.
An effective amount of the peptide-based body surface coloring
reagent for use in a skin care composition is a concentration of
about 0.01% to about 10%, preferably about 0.01% to about 5% by
weight relative to the total weight of the composition. This
proportion may vary as a function of the type of skin care
composition. Additionally, a mixture of different peptide-based
body surface coloring reagents having an affinity for different
pigments may be used in the composition. The peptide-based body
surface coloring reagents in the mixture need to be chosen so that
there is no interaction between the peptides that mitigates the
beneficial effect. Suitable mixtures of peptide-based body surface
coloring reagents may be determined by one skilled in the art using
routine experimentation. If a mixture of peptide-based body surface
coloring reagents is used in the composition, the total
concentration of the reagents is about 0.01% to about 10% by weight
relative to the total weight of the composition. The skin care
composition may further comprise at least one pigment, suitable
examples of which are given above. The concentration of the
peptide-based body surface coloring reagent in relation to the
concentration of the pigment may need to be optimized for best
results.
[0162] The composition may further comprise a cosmetically
acceptable medium for skin care compositions, examples of which are
described by Philippe et al. supra. For example, the cosmetically
acceptable medium may be an anhydrous composition containing a
fatty substance in a proportion generally of from about 10 to about
90% by weight relative to the total weight of the composition,
where the fatty phase contains at least one liquid, solid or
semi-solid fatty substance. The fatty substance includes, but is
not limited to, oils, waxes, gums, and so-called pasty fatty
substances. Alternatively, the compositions may be in the form of a
stable dispersion such as a water-in-oil or oil-in-water emulsion.
Additionally, the compositions may contain one or more conventional
cosmetic or dermatological additives or adjuvants including, but
not limited to, antioxidants, preserving agents, fillers,
surfactants, UVA and/or UVB sunscreens, fragrances, thickeners,
wetting agents and anionic, nonionic or amphoteric polymers, and
dyes.
[0163] Skin Coloring Compositions
[0164] In another embodiment, the peptide-based body surface
coloring reagent is a component of a skin coloring composition and
the peptide-based body surface coloring reagent comprises at least
one skin-binding peptide. The skin coloring composition comprises
one or more coloring agents. Any of the coloring agents described
above may be used.
[0165] The skin coloring compositions may be any cosmetic or
make-up product, including but not limited to foundations, blushes,
lipsticks, lip liners, lip glosses, eyeshadows and eyeliners. These
may be anhydrous make-up products comprising a cosmetically
acceptable medium which contains a fatty substance, or they may be
in the form of a stable dispersion such as a water-in-oil or
oil-in-water emulsion, as described above. In these compositions,
an effective amount of the peptide-based body surface coloring
reagent is generally from about 0.01% to about 40% by weight
relative to the total weight of the composition. Additionally, a
mixture of different peptide-based body surface coloring reagents
having an affinity for different pigments may be used in the
composition. The peptide-based body surface coloring reagents in
the mixture need to be chosen so that there is no interaction
between the peptides that mitigates the beneficial effect. Suitable
mixtures of peptide-based body surface coloring reagents may be
determined by one skilled in the art using routine experimentation.
If a mixture of peptide-based body surface coloring reagents is
used in the composition, the total concentration of the reagents is
about 0.01% to about 40% by weight relative to the total weight of
the composition.
[0166] Cosmetic Compositions
[0167] In another embodiment, the peptide-based body surface
coloring reagent is a component of a cosmetic composition and the
peptide-based body surface coloring reagent comprises at least one
hair binding peptide. Cosmetic compositions, as defined herein, are
compositions that may be applied to the eyelashes or eyebrows
including, but not limited to mascaras, and eyebrow pencils. These
cosmetic compositions comprise one or more coloring agents. Any of
the coloring agents described above may be used.
[0168] An effective amount of a peptide-based body surface coloring
reagent for use in a cosmetic composition is herein defined as a
proportion of from about 0.01% to about 20% by weight relative to
the total weight of the composition. Additionally, a mixture of
different peptide-based body surface coloring reagents having
affinity for different pigments may be used in the composition. The
peptide-based body surface coloring reagents in the mixture need to
be chosen so that there is no interaction between the peptides that
mitigates the beneficial effect. Suitable mixtures of peptide-based
body surface coloring reagents may be determined by one skilled in
the art using routine experimentation. If a mixture of
peptide-based body surface coloring reagents is used in the
composition, the total concentration of the reagents is about 0.01%
to about 20% by weight relative to the total weight of the
composition.
[0169] Cosmetic compositions may be anhydrous make-up products
comprising a cosmetically acceptable medium which contains a fatty
substance in a proportion generally of from about 10 to about 90%
by weight relative to the total weight of the composition, where
the fatty phase containing at least one liquid, solid or semi-solid
fatty substance, as described above. The fatty substance includes,
but is not limited to, oils, waxes, gums, and so-called pasty fatty
substances. Alternatively, these compositions may be in the form of
a stable dispersion such as a water-in-oil or oil-in-water
emulsion, as described above.
[0170] Nail Polish Compositions
[0171] In another embodiment, the peptide-based body surface
coloring reagent is a component of a nail polish composition and
the peptide-based body surface coloring reagent comprises at least
one nail-binding peptide. The nail polish compositions are used for
coloring fingernails and toenails and comprise one or more coloring
agents. Any of the coloring agents described above may be used.
[0172] An effective amount of a peptide-based body surface coloring
reagent for use in a nail polish composition is herein defined as a
proportion of from about 0.01% to about 20% by weight relative to
the total weight of the composition. Additionally, a mixture of
different peptide-based body surface coloring reagents having
affinity for different pigments may be used in the composition. The
peptide-based body surface coloring reagents in the mixture need to
be chosen so that there is no interaction between the peptides that
mitigates the beneficial effect. Suitable mixtures of peptide-based
body surface coloring reagents may be determined by one skilled in
the art using routine experimentation. If a mixture of
peptide-based body surface coloring reagents is used in the
composition, the total concentration of the reagents is about 0.01%
to about 20% by weight relative to the total weight of the
composition.
[0173] Components of a cosmetically acceptable medium for nail
polish compositions are described by Philippe et al. supra. The
nail polish composition typically contains a solvent and a film
forming substance, such as cellulose derivatives, polyvinyl
derivatives, acrylic polymers or copolymers, vinyl copolymers and
polyester polymers. Additionally, the nail polish may contain a
plasticizer, such as tricresyl phosphate, benzyl benzoate, tributyl
phosphate, butyl acetyl ricinoleate, triethyl citrate, tributyl
acetyl citrate, dibutyl phthalate or camphor.
[0174] Oral Care Compositions
[0175] In another embodiment, the peptide-based body surface
coloring reagent is a component of an oral care composition and the
peptide-based body surface coloring reagent comprises at least one
tooth-binding peptide. The oral care compositions of the invention
comprise at least one white colorant and are used to whiten teeth.
Suitable white colorants which may be used in the oral care
composition include, but are not limited to, white pigments such as
titanium dioxide and titanium dioxide nanoparticles; and white
minerals such as hydroxyapatite, and Zircon (zirconium
silicate).
[0176] The oral care compositions of the invention may be in the
form of powder, paste, gel, liquid, ointment, or tablet. Exemplary
oral care compositions include, but are not limited to, toothpaste,
dental cream, gel or tooth powder, mouth wash, breath freshener,
and dental floss. The oral care compositions comprise an effective
amount of the peptide-based body surface coloring reagent of the
invention in an orally acceptable carrier medium. An effective
amount of a peptide-based body surface coloring reagent for use in
an oral care composition may vary depending on the type of product.
Typically, the effective amount of the peptide-based body surface
coloring reagent is a proportion from about 0.01% to about 90% by
weight relative to the total weight of the composition.
Additionally, a mixture of different peptide-based body surface
coloring reagents having affinity for different pigments may be
used in the composition. The peptide-based body surface coloring
reagents in the mixture need to be chosen so that there is no
interaction between the peptides that mitigates the beneficial
effect. Suitable mixtures of peptide-based body surface coloring
reagents may be determined by one skilled in the art using routine
experimentation. If a mixture of peptide-based body surface
coloring reagents is used in the composition, the total
concentration of the reagents is about 0.001% to about 90% by
weight relative to the total weight of the composition.
[0177] Components of an orally acceptable carrier medium are
described by White et al. in U.S. Pat. No. 6,740,311; Lawler et al.
in U.S. Pat. No. 6,706,256; and Fuglsang et al. in U.S. Pat. No.
6,264,925; all of which are incorporated herein by reference. For
example, the oral care composition may comprise one or more of the
following: abrasives, surfactants, chelating agents, fluoride
sources, thickening agents, buffering agents, solvents, humectants,
carriers, bulking agents, and oral benefit agents, such as enzymes,
anti-plaque agents, anti-staining agents, anti-microbial agents,
anti-caries agents, flavoring agents, coolants, and salivating
agents.
Methods for Coloring a Body Surface
[0178] The peptide-based body surface coloring reagents of the
invention may be used in conjunction with one or more pigments to
color body surfaces, such as hair, skin, nails, and teeth. The body
surface-binding peptide block of the peptide-based body surface
coloring agent has an affinity for the body surface, while the
pigment-binding peptide block has an affinity for the pigment used.
The peptide-based body surface coloring reagent may be present in
the same composition as the pigment, or the peptide-based body
surface coloring reagent and the pigment may be present in two
different compositions. In one embodiment, a personal care
composition comprising at least one peptide-based body surface
coloring agent and at least one pigment is applied to a body
surface for a time sufficient for the peptide-based body surface
coloring agent, which is coupled to the pigment via the
pigment-binding peptide block, to bind to the body surface. In
another embodiment, at least one pigment is applied to a body
surface prior to the application of a composition comprising at
least one peptide-based body surface coloring reagent. In another
embodiment, a composition comprising at least one peptide-based
body surface coloring reagent is applied to the body surface prior
to the application of at least one pigment. In another embodiment,
at least one pigment and a composition comprising at least one
peptide-based body surface coloring reagent are applied to the body
surface concomitantly. Optionally, the composition comprising the
peptide-based body surface coloring reagent may be reapplied to the
body surface after the application of the pigment and the initial
application of the composition comprising the peptide-based body
surface coloring reagent. Additionally, a composition comprising a
polymeric sealant may be applied to the body surface after the
application of the pigment and the composition comprising a
peptide-based body surface coloring reagent.
[0179] Methods for Coloring Hair
[0180] The peptide-based body surface coloring reagents of the
invention may be used to attach a pigment to the surface of the
hair, thereby coloring the hair. The peptide-based body surface
coloring reagent and the pigment may be applied to the hair from
any suitable hair care composition, for example a hair colorant or
hair conditioner composition. These hair care compositions are well
known in the art and suitable compositions are described above.
[0181] In one embodiment, a pigment is applied to the hair for a
time sufficient for the pigment to bind to the hair, typically
between about 5 seconds to about 60 minutes. Optionally, the hair
may be rinsed to remove the pigment that has not bound to the hair.
Then, a composition comprising a peptide-based body surface
coloring reagent is applied to the hair for a time sufficient for
the body surface coloring reagent to bind to the hair and the
pigment, typically between about 5 seconds to about 60 minutes. The
composition comprising the peptide-based body surface coloring
reagent may be rinsed from the hair or left on the hair.
[0182] In another embodiment, a composition comprising a
peptide-based body surface reagent is applied to the hair for a
time sufficient for the hair-binding peptide block of the body
surface coloring reagent to bind to the hair, typically between
about 5 seconds to about 60 minutes. Optionally, the hair may be
rinsed to remove the composition that has not bound to the hair.
Then, a pigment is applied to the hair for a time sufficient for
the pigment to bind to the pigment-binding block of the body
surface coloring reagent, typically between about 5 seconds to
about 60 minutes. The unbound pigment may be rinsed from the hair
or left on the hair.
[0183] In another embodiment, a pigment and a composition
comprising a peptide-based body surface coloring reagent are
applied to the hair concomitantly for a time sufficient for the
body surface coloring reagent to bind to hair and the pigment,
typically between about 5 seconds to about 60 minutes. Optionally,
the hair may be rinsed to remove the unbound pigment and the
composition comprising a peptide-based body surface coloring
reagent from the hair.
[0184] In another embodiment, a pigment is provided as part of a
composition comprising a peptide-based body surface coloring
reagent, for example a hair coloring composition. The composition
comprising the pigment and the body surface coloring reagent is
applied to the hair for a time sufficient for the body surface
coloring reagent, which is coupled to the pigment through the
pigment-binding peptide block, to bind to the hair, typically
between about 5 seconds to about 60 minutes. The composition
comprising the pigment and the body surface coloring reagent may be
rinsed from the hair or left on the hair.
[0185] In any of the methods described above, the composition
comprising a peptide-based body surface coloring reagent may be
optionally reapplied to the hair after the application of the
pigment and the initial application of the composition comprising a
peptide-based body surface coloring reagent in order to further
enhance the durability of the colorant.
[0186] Additionally, in any of the methods described above, a
composition comprising a polymeric sealant may be optionally
applied to the hair after the application of the pigment and the
composition comprising a peptide-based body surface coloring
reagent in order to further enhance the durability of the colorant.
The composition comprising the polymeric sealant may be an aqueous
solution or a hair care composition, such as a conditioner or
rinse, comprising the polymeric sealant. Typically, the polymeric
sealant is present in the composition at a concentration of about
0.25% to about 10% by weight relative to the total weight of the
composition. Polymeric sealants are well know in the art of
personal care products and include, but are not limited to,
poly(allylamine), acrylates, acrylate copolymers, polyurethanes,
carbomers, methicones, amodimethicones, polyethylenene glycol,
beeswax, siloxanes, and the like. The choice of polymeric sealant
depends on the particular pigment and the peptide-based body
surface coloring reagent used. The optimum polymeric sealant may be
readily determined by one skilled in the art using routine
experimentation.
[0187] Methods for Coloring Skin
[0188] The peptide-based body surface coloring reagents of the
invention may be used to attach a pigment to the surface of the
skin, thereby coloring the skin. The peptide-based body surface
coloring reagent and the pigment may be applied to the skin from
any suitable skin care composition, for example a skin colorant or
skin conditioner composition. These skin care compositions are well
known in the art and suitable compositions are described above.
[0189] In one embodiment, a pigment is applied to the skin for a
time sufficient for the pigment to bind to the skin, typically
between about 5 seconds to about 60 minutes. Optionally, the skin
may be rinsed to remove the pigment that has not bound to the skin.
Then, a composition comprising a peptide-based body surface
coloring reagent is applied to the skin for a time sufficient for
the body surface coloring reagent to bind to the skin and the
pigment, typically between about 5 seconds to about 60 minutes. The
composition comprising the peptide-based body surface coloring
reagent may be rinsed from the skin or left on the skin.
[0190] In another embodiment, a composition comprising a
peptide-based body surface coloring reagent is applied to the skin
for a time sufficient for the skin-binding peptide block of the
body surface coloring reagent to bind to the skin, typically
between about 5 seconds to about 60 minutes. Optionally, the skin
may be rinsed to remove the composition that has not bound to the
skin. Then, a pigment is applied to the skin for a time sufficient
for the pigment to bind to the pigment-binding block of the body
surface coloring reagent, typically between about 5 seconds to
about 60 minutes. The unbound pigment may be rinsed from the skin
or left on the skin.
[0191] In another embodiment, a pigment and a composition
comprising a peptide-based body surface coloring reagent are
applied to the skin concomitantly for a time sufficient for the
body surface coloring reagent to bind to skin and the pigment,
typically between about 5 seconds to about 60 minutes. Optionally,
the skin may be rinsed to remove the unbound pigment and the
composition comprising a peptide-based body surface coloring
reagent from the skin.
[0192] In another embodiment, a pigment is provided as part of the
composition comprising a peptide-based body surface coloring
reagent, for example a skin coloring composition. The composition
comprising the pigment and the body surface coloring reagent is
applied to the skin for a time sufficient for the body surface
coloring reagent, which is coupled to the pigment through the
pigment-binding block, to bind to the skin, typically between about
5 seconds to about 60 minutes. The composition comprising the
pigment and the body surface coloring reagent may be rinsed from
the skin or left on the skin.
[0193] In any of the methods described above, the composition
comprising a peptide-based body surface coloring reagent may be
optionally reapplied to the skin after the application of the
pigment and the initial application of the composition comprising a
peptide-based body surface coloring reagent in order to further
enhance the durability of the colorant.
[0194] Additionally, in any of the methods described above, a
composition comprising a polymeric sealant may be optionally
applied to the skin after the application of the pigment and the
composition comprising a peptide-based body surface coloring
reagent in order to further enhance the durability of the colorant.
Any of the polymeric sealants described above for hair coloring may
be used in the form of an aqueous solution or a skin care
composition.
[0195] Methods for Coloring Nails, Eyebrows, Eyelashes, and
Teeth
[0196] The methods described above for coloring hair and skin may
also be applied to coloring finger nails and toenails, eyebrows,
eyelashes, and teeth by applying the appropriate composition,
specifically, a nail polish composition, a cosmetic composition, or
an oral care composition, to the body surface of interest.
EXAMPLES
[0197] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various uses and conditions.
[0198] The meaning of abbreviations used is as follows: "min" means
minute(s), "sec" means second(s), "h" means hour(s), ".mu.L" means
microliter(s), "mL" means milliliter(s), "L" means liter(s), "nm"
means nanometer(s), "mm" means millimeter(s), "cm" means
centimeter(s), ".mu.m" means micrometer(s), "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), ".mu.mole" means
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "mg"
means milligram(s), "g" means the gravitation constant, "rpm" means
revolutions per minute, "pfu" means plague forming unit, "BSA"
means bovine serum albumin, "ELISA" means enzyme linked
immunosorbent assay, "IPTG" means isopropyl
.beta.-D-thiogalactopyranoside, "A" means absorbance, "A.sub.450"
means the absorbance measured at a wavelength of 450 nm,
"OD.sub.600" means the optical density measured at 600 nanometers,
"TBS" means Tris-buffered saline, "TBST-X" means Tris-buffered
saline containing Tween.RTM. 20 where "X" is the weight percent of
Tween.RTM. 20, "Xgal" means
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, "SEM" means
standard error of the mean, "ESCA" means electron spectroscopy for
chemical analysis, "eV" means electron volt(s), "TGA" means
thermogravimetric analysis, "GPC" means gel permeation
chromatography, "MW" means molecular weight, "Mw" means
weight-average molecular weight, "vol %" means volume percent, "wt
%" means weight percent, "NMR" means nuclear magnetic resonance
spectroscopy, "MALDI mass spectrometry" means matrix assisted,
laser desorption ionization mass spectrometry, "atm" means
atmosphere(s), "kPa" means kilopascal(s), "SLPM" means standard
liter per minute, "psi" means pounds per square inch, "RCF" means
relative centrifugal field.
[0199] General Methods:
[0200] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W.
Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience, N.Y., 1987.
[0201] Materials and methods suitable for the maintenance and
growth of bacterial cultures are also well known in the art.
Techniques suitable for use in the following Examples may be found
in Manual of Methods for General Bacteriology, Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society
for Microbiology, Washington, D.C., 1994, or by Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. All
reagents, restriction enzymes and materials used for the growth and
maintenance of bacterial cells were obtained from Aldrich Chemicals
(Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life
Technologies (Rockville, Md.), or Sigma Chemical Company (St.
Louis, Mo.), unless otherwise specified.
Example 1
Selection of Hair-Binding Phage Peptides Using Standard
Biopanning
[0202] The purpose of this Example was to identify hair-binding
phage peptides that bind to normal hair and to bleached hair using
standard phage display biopanning.
Phage Display Peptide Libraries:
[0203] The phage libraries used in the present invention,
Ph.D.-12.TM. Phage Display Peptide Library Kit and Ph.D.-7.TM.
Phage Display Library Kit, were purchased from New England BioLabs
(Beverly, Mass.). These kits are based on a combinatorial library
of random peptide 7 or 12-mers fused to a minor coat protein (pill)
of M13 phage. The displayed peptide is expressed at the N-terminus
of pill, such that after the signal peptide is cleaved, the first
residue of the coat protein is the first residue of the displayed
peptide. The Ph.D.-7 and Ph.D.-12 libraries consist of
approximately 2.8.times.10.sup.9 and 2.7.times.10.sup.9 sequences,
respectively. A volume of 10 .mu.L contains about 55 copies of each
peptide sequence. Each initial round of experiments was carried out
using the original library provided by the manufacturer in order to
avoid introducing any bias into the results.
Preparation of Hair Samples:
[0204] The samples used as normal hair were 6-inch medium brown
human hairs obtained from International Hair Importers and Products
(Bellerose, N.Y.). The hairs were placed in 90% isopropanol for 30
min at room temperature and then washed 5 times for 10 min each
with deionized water. The hairs were air-dried overnight at room
temperature.
[0205] To prepare the bleached hair samples, the medium brown human
hairs were placed in 6% H.sub.2O.sub.2, which was adjusted to pH
10.2 with ammonium hydroxide, for 10 min at room temperature and
then washed 5 times for 10 min each with deionized water. The hairs
were air-dried overnight at room temperature.
[0206] The normal and bleached hair samples were cut into 0.5 to 1
cm lengths and about 5 to 10 mg of the hairs was placed into wells
of a custom 24-well biopanning apparatus that had a pig skin
bottom. An equal number of the pig skin bottom wells were left
empty. The pig skin bottom apparatus was used as a subtractive
procedure to remove phage-peptides that have an affinity for skin.
This apparatus was created by modifying a dot blot apparatus
(obtained from Schleicher & Schuell, Keene, N.H.) to fit the
biopanning process. Specifically, the top 96-well block of the dot
blot apparatus was replaced by a 24-well block. A 4.times.6 inch
treated pig skin was placed under the 24-well block and panning
wells with a pig skin bottom were formed by tightening the
apparatus. The pig skin was purchased from a local supermarket and
stored at -80.degree. C. Before use, the skin was placed in
deionized water to thaw, and then blotted dry using a paper towel.
The surface of the skin was wiped with 90% isopropanol, and then
rinsed with deionized water. The 24-well apparatus was filled with
blocking buffer consisting of 1 mg/mL BSA in TBST containing 0.5%
Tween.RTM. 20 (TBST-0.5%) and incubated for 1 h at 4.degree. C. The
wells and hairs were washed 5 times with TBST-0.5%. One milliliter
of TBST-0.5% containing 1 mg/mL BSA was added to each well. Then,
10 .mu.L of the original phage library (2.times.10.sup.11 pfu),
either the 12-mer or 7-mer library, was added to the pig skin
bottom wells that did not contain a hair sample and the phage
library was incubated for 15 min at room temperature. The unbound
phages were then transferred to pig skin bottom wells containing
the hair samples and were incubated for 15 min at room temperature.
The hair samples and the wells were washed 10 times with TBST-0.5%.
The hairs were then transferred to clean, plastic bottom wells of a
24-well plate and 1 mL of a non-specific elution buffer consisting
of 1 mg/mL BSA in 0.2 M glycine-HCl, pH 2.2, was added to each well
and incubated for 10 min to elute the bound phages. Then, 160 .mu.L
of neutralization buffer consisting of 1 M Tris-HCl, pH 9.2, was
added to each well. The eluted phages from each well were
transferred to a new tube for titering and sequencing.
[0207] To titer the bound phages, the eluted phage was diluted with
SM buffer (100 mM NaCl, 12.3 mM MgSO.sub.4-7H.sub.2O, 50 mM
Tris-HCl, pH 7.5, and 0.01 wt/vol % gelatin) to prepare 10-fold
serial dilutions of 10.sup.1 to 10.sup.4. A 10 .mu.L aliquot of
each dilution was incubated with 200 .mu.L of mid-log phase E. coli
ER2738 (New England BioLabs), grown in LB medium for 20 min and
then mixed with 3 mL of agarose top (LB medium with 5 mM
MgCl.sub.2, and 0.7% agarose) at 45.degree. C. This mixture was
spread onto a S-Gal.TM./LB agar plate (Sigma Chemical Co.) and
incubated overnight at 37.degree. C. The S-Gal.TM./LB agar blend
contained 5 g of tryptone, 2.5 g of yeast extract, 5 g of sodium
chloride, 6 g of agar, 150 mg of
3,4-cyclohexenoesculetin-.beta.-D-galactopyranoside (S-Gal.TM.),
250 mg of ferric ammonium citrate and 15 mg of isopropyl
.beta.-D-thiogalactoside (IPTG) in 500 mL of distilled water. The
plates were prepared by autoclaving the S-Gal.TM./LB for 15 to 20
min at 121-124.degree. C. The single black plaques were randomly
picked for DNA isolation and sequence analysis.
[0208] The remaining eluted phages were amplified by incubating
with diluted E. coli ER2738, from an overnight culture diluted
1:100 in LB medium, at 37.degree. C. for 4.5 h. After this time,
the cell culture was centrifuged for 30 s and the upper 80% of the
supernatant was transferred to a fresh tube, 1/6 volume of PEG/NaCl
(20% polyethylene glyco-800, 2.5 M sodium chloride) was added, and
the phage was allowed to precipitate overnight at 4.degree. C. The
precipitate was collected by centrifugation at 10,000.times.g at
4.degree. C. and the resulting pellet was resuspended in 1 mL of
TBS. This was the first round of amplified stock. The amplified
first round phage stock was then titered according to the same
method as described above. For the next round of biopanning, more
than 2.times.10.sup.11 pfu of phage stock from the first round was
used. The biopanning process was repeated for 3 to 6 rounds
depending on the experiments.
[0209] The single plaque lysates were prepared following the
manufacture's instructions (New England Biolabs) and the single
stranded phage genomic DNA was purified using the QIAprep Spin M13
Kit (Qiagen, Valencia, Calif.) and sequenced at the DuPont
Sequencing Facility using -96 gill sequencing primer
(5'-CCCTCATAGTTAGCGTAACG-3'), given as SEQ ID NO:62. The displayed
peptide is located immediately after the signal peptide of gene
III.
[0210] The amino acid sequences of the eluted normal hair-binding
phage peptides from the 12-mer library isolated from the fifth
round of biopanning are given in Table 1. The amino acid sequences
of the eluted bleached hair-binding phage peptides from the 12-mer
library isolated from the fifth round of biopanning are given in
Table 2. Repeated amino acid sequences of the eluted normal
hair-binding phage peptides from the 7-mer library from 95 randomly
selected clones, isolated from the third round of biopanning, are
given in Table 3. TABLE-US-00002 TABLE 1 Amino Acid Sequences of
Eluted Normal Hair- Binding Phage Peptides from 12-Mer Library
Amino Acid Clone ID Sequence SEQ ID NO: Frequency.sup.1 1
RVPNKTVTVDGA 5 5 2 DRHKSKYSSTKS 6 2 3 KNFPQQKEFPLS 7 2 4
QRNSPPAMSRRD 8 2 5 TRKPNMPHGQYL 9 2 6 KPPHLAKLPFTT 10 1 7
NKRPPTSHRIHA 11 1 8 NLPRYQPPCKPL 12 1 9 RPPWKKPIPPSE 13 1 10
RQRPKDHFFSRP 14 1 11 SVPNKXVTVDGX 15 1 12 TTKWRHRAPVSP 16 1 13
WLGKNRIKPRAS 17 1 14 SNFKTPLPLTQS 18 1 15 SVSVGMKPSPRP 3 1
.sup.1The frequency represents the number of identical sequences
that occurred out of 23 sequenced clones.
[0211] TABLE-US-00003 TABLE 2 Amino Acid Sequences of Eluted
Bleached Hair- Binding Phage Peptides from 12-Mer Library Amino
Acid Clone ID Sequence SEQ ID NO: Frequency.sup.1 1 KELQTRNVVQRE 19
8 2 QRNSPPAMSRRD 8 5 3 TPTANQFTQSVP 20 2 4 AAGLSQKHERNR 21 2 5
ETVHQTPLSDRP 22 1 6 KNFPQQKEFPLS 7 1 7 LPALHIQRHPRM 23 1 8
QPSHSQSHNLRS 24 1 9 RGSQKSKPPRPP 25 1 10 THTQKTPLLYYH 26 1 11
TKGSSQAILKST 27 1 .sup.1The frequency represents the number of
identical sequences that occurred out of 24 sequenced clones.
[0212] TABLE-US-00004 TABLE 3 Amino Acid Sequences of Eluted Normal
Hair- Binding Phage Peptides from 7-Mer Library Clone ID Amino Acid
Sequence SEQ ID NO: A DLHTVYH 28 B HIKPPTR 29 D HPVWPAI 30 F
MPLYYLQ 31 F.sup.1 HLTVPWRGGGSAVPFYSHSQITLPNH 32 G.sup.1
GPHDTSSGGVRPNLHHTSKKEKREN 33 RKVPFYSHSVTSRGNV H KHPTYRQ 34 I
HPMSAPR 35 J MPKYYLQ 36 .sup.1There was a multiple DNA fragment
intersion in these clones.
Example 2
Selection of High Affinity Hair-Binding Phage Peptides Using a
Modified Method
[0213] The purpose of this Example was to identify hair-binding
phage peptides with a higher binding affinity.
[0214] The hairs that were treated with the acidic elution buffer,
as described in Example 1, were washed three more times with the
elution buffer and then washed three times with TBST-0.5%. These
hairs, which had acid resistant phage peptides still attached, were
used to directly infect 500 .mu.L of mid-log phase bacterial host
cells, E. coli ER2738 (New England BioLabs), which were then grown
in LB medium for 20 min and then mixed with 3 mL of agarose top (LB
medium with 5 mM MgCl.sub.2, and 0.7% agarose) at 45.degree. C.
This mixture was spread onto a LB medium/IPTG/S-Gal.TM. plate (LB
medium with 15 g/L agar, 0.05 g/L IPTG, and 0.04 g/L S-Gal.TM.) and
incubated overnight at 37.degree. C. The black plaques were counted
to calculate the phage titer. The single black plaques were
randomly picked for DNA isolation and sequencing analysis, as
described in Example 1. This process was performed on the normal
and bleached hair samples that were screened with the 7-mer and
12-mer phage display libraries, as described in Example 1. The
amino acid sequences of these high affinity, hair-binding phage
peptides are given in Tables 4-7. TABLE-US-00005 TABLE 4 Amino Acid
Sequences of High Affinity, Normal Hair-Binding Phage Peptides from
7-Mer Library Clone ID Amino Acid Sequence SEQ ID NO: D5
GPHDTSSGGVRPNL 33 HHTSKKEKRENRKVP FYSHSVTSRGNV.sup.1 A36 MHAHSIA 37
B41 TAATTSP 38 .sup.1There was a multiple DNA fragment intersion in
this clone.
[0215] TABLE-US-00006 TABLE 5 Amino Acid Sequences of High
Affinity, Bleached Hair-Binding Phage Peptides from 7-Mer Library
Clone ID Amino Acid Sequence SEQ ID NO: D39 LGIPQNL 39 B1 TAATTSP
38
[0216] TABLE-US-00007 TABLE 6 Amino Acid Sequences of High
Affinity, Normal Hair-Binding Phage Peptides from 12-Mer Library
Clone ID Amino Acid Sequence SEQ ID NO: C2 AKPISQHLQRGS 40 A3
APPTPAAASATT 41 F9 DPTEGARRTIMT 42 A19 EQISGSLVAAPW 43 F4
LDTSFPPVPFHA 44 F35 LPRIANTWSPS 45 D21 RTNAADHPAAVT 46 C10
SLNWVTIPGPKI 47 C5 TDMQAPTKSYSN 48 D20 TIMTKSPSLSCG 49 C18
TPALDGLRQPLR 50 A20 TYPASRLPLLAP 51 C13 AKTHKHPAPSYS 52 G-D20
YPSFSPTYRPAF 53 A23 TDPTPFSISPER 54 F67 SQNWQDSTSYSN 55 F91
WHDKPQNSSKST 56 G-F1 LDVESYKGTSMP 4
[0217] TABLE-US-00008 TABLE 7 Amino Acid Sequences of High
Affinity, Bleached Hair-Binding Phage Peptides from 12-Mer Library
Clone ID Amino Acid Sequence SEQ ID NO: A5 EQISGSLVAAPW 43 C4
NEVPARNAPWLV 57 D30 NSPGYQADSVAIG 58 C44 AKPISQHLQRGS 40 E66
LDTSFPPVPFHA 44 C45 SLNWVTIPGPKI 47 E18 TQDSAQKSPSPL 59
Example 3
Selection of High Affinity Fingernail-Binding Phage Peptides
[0218] The purpose of this Example was to identify phage peptides
that have a high binding affinity to fingernails. The modified
biopanning method described in Example 2 was used to identify high
affinity, fingernail-binding phage-peptide clones.
[0219] Human fingernails were collected from test subjects. The
fingernails were cleaned by brushing with soap solution, rinsed
with deionized water, and allowed to air-dry at room temperature.
The fingernails were then powdered under liquid N.sub.2, and 10 mg
of the fingernails was added to each well of a 96-well filter
plate. The fingernail samples were treated for 1 h with blocking
buffer consisting of 1 mg/mL BSA in TBST-0.5%, and then washed with
TBST-0.5%. The fingernail samples were incubated with phage library
(Ph.D-12 Phage Display Peptide Library Kit), and washed 10 times
using the same conditions described in Example 1. After the acidic
elution step, described in Example 1, the fingernail samples were
washed three more times with the elution buffer and then washed
three times with TBST-0.5%. The acid-treated fingernails, which had
acid resistant phage peptides still attached, were used to directly
infect E. coli ER2738 cells as described in Example 2. This
biopanning process was repeated three times. A total of 75 single
black phage plaques were picked randomly for DNA isolation and
sequencing analysis and two repeated clones were identified. The
amino acid sequences of these phage peptides are listed in Table 8.
These fingernail binding peptides were also found to bind well to
bleached hair. TABLE-US-00009 TABLE 8 Amino Acid Sequences of High
Affinity Fingernail-Binding Phage Peptides Amino Acid Clone ID
Sequence SEQ ID NO: Frequency.sup.1 F01 ALPRIANTWSPS 60 15 D05
YPSFSPTYRPAF 53 26 .sup.1The frequency represents the number of
identical sequences that occurred out of 75 sequenced clones.
Example 4
Selection of High Affinity Skin-Binding Phage Peptides
[0220] The purpose of this Example was to identify phage peptides
that have a high binding affinity to skin. The modified biopanning
method described in Examples 2 and 4 was used to identify the high
affinity, skin-binding phage-peptide clones. Pig skin served as a
model for human skin in the process.
[0221] The pig skin was prepared as described in Example 1. Three
rounds of screenings were performed with the custom, pig skin
bottom biopanning apparatus using the same procedure described in
Example 4. A total of 28 single black phage plaques were picked
randomly for DNA isolation and sequencing analysis and one repeated
clone was identified. The amino acid sequence of this phage
peptide, which appeared 9 times out of the 28 sequences, was
TPFHSPENAPGS, given as SEQ ID NO:61.
Example 5
Quantitative Characterization of the Binding Affinity of
Hair-Binding Phage Clones
[0222] The purpose of this Example was to quantify the binding
affinity of phage clones by titering and ELISA.
Titering of Hair-Binding Phage Clones:
[0223] Phage clones displaying specific peptides were used for
comparing the binding characteristics of different peptide
sequences. A titer-based assay was used to quantify the phage
binding. This assay measures the output pfu retained by 10 mg of
hair surfaces, having a signal to noise ratio of 10.sup.3 to
10.sup.4. The input for all the phage clones was 10.sup.14 pfu. It
should be emphasized that this assay measures the
peptide-expressing phage particle, rather than peptide binding.
[0224] Normal hairs were cut into 0.5 cm lengths and 10 mg of the
cut hair was placed in each well of a 96-well filter plate
(Qiagen). Then, the wells were filled with blocking buffer
containing 1 mg/mL BSA in TBST-0.5% and incubated for 1 h at
4.degree. C. The hairs were washed 5 times with TBST-0.5%. The
wells were then filled with 1 mL of TBST-0.5% containing 1 mg/mL
BSA and then purified phage clones (10.sup.14 pfu) were added to
each well. The hair samples were incubated for 15 min at room
temperature and then washed 10 times with TBST-0.5%. The hairs were
transferred to a clean well and 1.0 mL of a non-specific elution
buffer, consisting of 1 mg/mL BSA in 0.2 M Glycine-HCl at pH 2.2,
was added to each well. The samples were incubated for 10 min and
then 160 .mu.L of neutralization buffer (1 M Tris-HCl, pH 9.2) was
added to each well. The eluted phages from each well were
transferred to a new tube for titering and sequencing analysis.
[0225] To titer the bound phages, the eluted phage was diluted with
SM buffer to prepare 10-fold serial dilutions of 10.sup.1 to
10.sup.8. A 10 .mu.L aliquot of each dilution was incubated with
200 .mu.L of mid-log phase E. coli ER2738 (New England BioLabs),
and grown in LB medium for 20 min and then mixed with 3 mL of
agarose top (LB medium with 5 mM MgCl.sub.2, and 0.7% agarose) at
45.degree. C. This mixture was spread onto a LB medium/IPTG/Xgal
plate (LB medium with 15 g/L agar, 0.05 g/L IPTG, and 0.04 g/L
Xgal) and incubated overnight at 37.degree. C. The blue plaques
were counted to calculate the phage titers, which are given in
Table 9. TABLE-US-00010 TABLE 9 Titer of Hair-Binding Phage Clones
Clone ID SEQ ID NO: Phage Titer A 28 7.50 .times. 10.sup.4 B 29
1.21 .times. 10.sup.5 D 30 8.20 .times. 10.sup.4 E 31 1.70 .times.
10.sup.5 F 32 1.11 .times. 10.sup.6 G 33 1.67 .times. 10.sup.8 H 34
1.30 .times. 10.sup.6 1 35 1.17 .times. 10.sup.6 J 36 1.24 .times.
10.sup.6
Characterization of Hair-Binding Phage Clones by ELISA:
[0226] Enzyme-linked immunosorbent assay (ELISA) was used to
evaluate the hair-binding specificity of selected phage-peptide
clones. Phage-peptide clones identified in Examples 1 and 2 along
with a randomly chosen control G-F9, KHGPDLLRSAPR (given as SEQ ID
NO:63) were amplified. More than 10.sup.14 pfu phages were added to
pre-blocked hair surfaces. The same amount of phages was also added
to pre-blocked pig skin surfaces as a control to demonstrate the
hair-binding specificity.
[0227] A unique hair or pig skin-bottom 96-well apparatus was
created by applying one layer of Parafilm.RTM. under the top
96-well block of a Minifold I Dot-Blot System (Schleicher &
Schuell, Inc., Keene, N.H.), adding hair or a layer of hairless pig
skin on top of the Parafilm.RTM. cover, and then tightening the
apparatus. For each clone to be tested, the hair-covered well was
incubated for 1 h at room temperature with 200 .mu.L of blocking
buffer, consisting of 2% non-fat dry milk (Schleicher &
Schuell, Inc.) in TBS. A second Minifold system with pig skin at
the bottom of the wells was treated with blocking buffer
simultaneously to serve as a control. The blocking buffer was
removed by inverting the systems and blotting them dry with paper
towels. The systems were rinsed 6 times with wash buffer consisting
of TBST-0.05%. The wells were filled with 200 .mu.L of TBST-0.5%
containing 1 mg/mL BSA and then 10 .mu.L (over 10.sup.12 copies) of
purified phage stock was added to each well. The samples were
incubated at 37.degree. C. for 15 min with slow shaking. The
non-binding phage was removed by washing the wells 10 to 20 times
with TBST-0.5%. Then, 100 .mu.L of horseradish peroxidase/anti-M13
antibody conjugate (Amersham USA, Piscataway, N.J.), diluted 1:500
in the blocking buffer, was added to each well and incubated for 1
h at room temperature. The conjugate solution was removed and the
wells were washed 6 times with TBST-0.05%. TMB substrate (200
.mu.L), obtained from Pierce Biotechnology (Rockford, Ill.) was
added to each well and the color was allowed to develop for between
5 to 30 min, typically for 10 min, at room temperature. Then, stop
solution (200 .mu.L of 2 M H.sub.2SO.sub.4) was added to each well
and the solution was transferred to a 96-well plate and the
A.sub.450 was measured using a microplate spectrophotometer
(Molecular Devices, Sunnyvale, Calif.). The resulting absorbance
values, reported as the mean of at least three replicates, and the
standard error of the mean (SEM) are given in Table 10.
TABLE-US-00011 TABLE 10 Results of ELISA Assay with Skin and Hair
SEQ ID Hair Pig Skin Clone ID NO: A.sub.450 SEM A.sub.450 SEM G-F9
63 0.074 0.057 -0.137 0.015 (Control) D21 46 1.051 0.16 0.04 0.021
D39 39 0.685 0.136 0.086 0.019 D5 33 0.652 0.222 0.104 0.023 A36 37
0.585 0.222 0.173 0.029 C5 48 0.548 0.263 0.047 0.037 C10 47 0.542
0.105 0.032 0.012 A5 43 0.431 0.107 0.256 0.022 B1 38 0.42 0.152
0.127 0.023 D30 58 0.414 0.119 0.287 0.045 C13 52 0.375 0.117 0.024
0.016 C18 50 0.34 0.197 0.132 0.023
[0228] As can be seen from the data in Table 10, all the
hair-binding clones had a significantly higher binding affinity for
hair than the control. Moreover, the hair-binding clones exhibited
various degrees of selectivity for hair compared to pig skin. Clone
D21 had the highest selectivity for hair, having a very strong
affinity for hair and a very low affinity for pig skin.
Example 6
Confirmation of Peptide Binding Specificity and Affinity
[0229] The purpose of this Example was to test the peptide binding
site specificity and affinity of the hair-binding peptide D21 using
a competition ELISA. The ELISA assay only detects phage particles
that remain bound to the hair surface. Therefore, if the synthetic
peptide competes with the phage particle for the same binding site
on hair surface, the addition of the synthetic peptide into the
ELISA system will significantly reduce the ELISA results due to the
peptide competition.
[0230] The synthetic hair-binding peptide D21, given as SEQ ID
NO:46, was synthesized by SynPep (Dublin, Calif.). As a control, an
unrelated synthetic skin-binding peptide, given as SEQ ID NO:61,
was added to the system. The experimental conditions were similar
to those used in the ELISA method described in Example 5. Briefly,
100 .mu.L of Binding Buffer (1.times.TBS with 0.1% Tween.RTM.20 and
1 mg/mL BSA) and 10.sup.11 pfu of the pure D21 phage particles were
added to each well of the 96-well filter plate, which contained a
sample of normal hair. The synthetic peptide (100 .mu.g) was added
to each well (corresponding to concentration of 0.8 mM). The
reactions were carried out at room temperature for 1 h with gentle
shaking, followed by five washes with TBST-0.5%. The remaining
steps were identical to the those used in the ELISA method
described in Example 5. The ELISA results, presented as the
absorbance at 450 nm (A.sub.450), are shown in Table 11. Each
individual ELISA test was performed in triplicate; the values in
Table 11 are the means of the triplicate determinations.
TABLE-US-00012 TABLE 11 Results of Peptide Competition ELISA Sample
A.sub.450 SEM Antibody-Conjugate 0.199 0.031 Phage D21 1.878 0.104
Phage D21 and D21 1.022 0.204 Peptide Phage D21 and 2.141 0.083
Control Peptide
[0231] These results demonstrated that the synthetic peptide D21
does compete with the phage clone D21 for the same binding sites on
the hair surface.
Example 7
Selection of Shampoo-Resistant Hair-Binding Phage-Peptides Using
Biopanning
[0232] The purpose of this Example was to select shampoo-resistant
hair-binding phage-peptides using biopanning with shampoo
washes.
[0233] In order to select shampoo-resistant hair-binding peptides,
a biopanning experiment using 12-mer phage peptide libraries
against normal and bleached hairs was performed, as described in
Example 2. Instead of using normal TBST buffer to wash-off the
unbounded phages, the phage-complexed hairs were washed with 10%,
30% and 50% shampoo solutions (Pantene Pro-V shampoo, Sheer Volume,
Proctor & Gamble, Cincinnati, Ohio), for 5 min in separate
tubes, followed by six TBS buffer washes. The washed hairs were
directly used to infect host bacterial cells as described in the
modified biopanning method, described in Example 2.
[0234] A potential problem with this method is the effect of the
shampoo on the phage's ability to infect bacterial host cells. In a
control experiment, a known amount of phage particles was added to
a 10% shampoo solution for 5 min, and then a portion of the
solution was used to infect bacterial cells. The titer of the
shampoo-treated phage was 90% lower than that of the untreated
phage. The 30% and 50% shampoo treatments gave even more severe
damage to the phage's ability to infect host cells. Nevertheless,
two shampoo-resistant hair-binding phage-peptides were identified,
as shown in Table 12. TABLE-US-00013 TABLE 12 Peptide Sequences of
Shampoo-Resistant Hair- binding Phage Peptides Identified Using the
Biopanning Method Clone Sequence Target SEQ ID NO: I-B5
TPPELLHGDPRS Normal and 66 Bleached Hair H-B1 TPPTNVLMLATK Normal
Hair 69
Example 8
Selection of Shampoo-Resistant Hair-Binding Phage-Peptides Using
PCR
[0235] The purpose of this Example was to select shampoo-resistant
hair-binding phage-peptides using a PCR method to avoid the problem
of shampoo induced damage to the phage. This principle of the PCR
method is that DNA fragments inside the phage particle can be
recovered using PCR, regardless of the phage's viability, and that
the recovered DNA fragments, corresponding to the hair-binding
peptide sequences, can then been cloned back into a phage vector
and packaged into healthy phage particles.
[0236] Biopanning experiments were performed using 7-mer and 12-mer
phage-peptide libraries against normal and bleached hairs, as
described in Example 1. After the final wash, the phage-treated
hairs were subjected to 5 min of shampoo washes, followed by six
TBS buffer washes. The shampoo-washed hairs were put into a new
tube filled with 1 mL of water, and boiled for 15 min to release
the DNA. This DNA-containing, boiled solution was used as a DNA
template for PCR reactions. The primers used in the PCR reaction
were primers: M13KE-1412 Forward 5'-CAAGCCTCAGCGACCGAATA-3', given
as SEQ ID NO:67 and M13KE-1794 Reverse
5'-CGTAACACTGAGTTTCGTCACCA-3', given SEQ ID NO:68. The PCR
conditions were: 3 min denaturing at 96.degree. C., followed by 35
cycles of 94.degree. C. for 30 sec, 50.degree. C. for 30 sec and
60.degree. C. for 2 min. The PCR products (.about.400 bp), and
M13KE vector (New England BioLabs) were digested with restriction
enzymes Eag I and Acc65 I. The ligation and transformation
conditions, as described in the Ph.D..TM. Peptide Display Cloning
System (New England Biolabs), were used. The amino acid sequence of
the resulting shampoo-resistant hair-binding phage-peptide is
NTSQLST, given as SEQ ID NO:70.
Example 9
Determination of the Affinity of Hair-Binding and Skin-Binding
Peptides
[0237] The purpose of this Example was to determine the affinity of
the hair-binding and skin-binding peptides for their respective
substrates, measured as MB.sub.50 values, using an ELISA assay.
[0238] Hair-binding and skin-binding peptides were synthesized by
SynPep Inc. (Dublin, Calif.). The peptides were biotinylated by
adding a biotinylated lysine residue at the C-terminus of the amino
acid binding sequences for detection purposes and an amidated
cysteine was added to the C-terminus of the sequence. The amino
acid sequences of the peptides tested are given as SEQ ID
NOs:71-74, as shown in Table 13.
[0239] For hair samples, the procedure used was as follows. The
setup of the surface specific 96-well system used was the same as
that described in Example 5. Briefly, the 96-wells with hair or pig
skin surfaces were blocked with blocking buffer (SuperBlock.TM.
from Pierce Chemical Co., Rockford, Ill.) at room temperature for 1
h, followed by six washes with TBST-0.5%, 2 min each, at room
temperature. Various concentrations of biotinylated, binding
peptide were added to each well, incubated for 15 min at 37.degree.
C., and washed six times with TBST-0.5%, 2 min each, at room
temperature. Then, streptavidin-horseradish peroxidase (HRP)
conjugate (Pierce Chemical Co.) was added to each well (1.0 .mu.g
per well), and incubated for 1 h at room temperature. After the
incubation, the wells were washed six times with TBST-0.5%, 2 min
each at room temperature. Finally, the color development and the
measurement were performed as described in Example 5.
[0240] For the measurement of MB.sub.50 of the peptide-skin
complexes, the following procedure was used. First, the pigskin was
treated to block the endogenous biotin in the skin. This was done
by adding streptavidin to the blocking buffer. After blocking the
pigskin sample, the skin was treated with D-biotin to block the
excess streptavidin binding sites. The remaining steps were
identical to those used for the hair samples.
[0241] The results were plotted as A.sub.450 versus the
concentration of peptide using GraphPad Prism 4.0 (GraphPad
Software, Inc., San Diego, Calif.). The MB.sub.50 values were
calculated from Scatchard plots and are summarized in Table 13. The
results demonstrate that the binding affinity of the hair-binding
peptides (D21, F35, and I-B5) and the skin binding peptide (SEQW ID
NO:61) for their respective substrate was high, while the binding
affinity of the hair-binding peptides (D-21 and I-B5) for skin was
relatively low. TABLE-US-00014 TABLE 13 Summary of MB.sub.50 Values
for Hair and Skin-Binding Peptides Peptide Sequence Binding Peptide
Tested* Substrate MB.sub.50, M D21 SEQ ID NO: 71 Normal Hair 2
.times. 10.sup.-6 F35 SEQ ID NO: 72 Bleached Hair 3 .times.
10.sup.-6 I-B5 SEQ ID NO: 73 Normal and 3 .times. 10.sup.-7
Bleached Hair D21 SEQ ID NO: 71 Pig Skin 4 .times. 10.sup.-5 I-B5
SEQ ID NO: 73 Pig Skin >1 .times. 10.sup.-4 SEQ ID NO: 61 SEQ ID
NO: 74 Pig Skin 7 .times. 10.sup.-7 *The peptides tested were
biotinylated at the C-terminus of the amino acid binding sequences
and an amidated cysteine was added to the C-terminus of the binding
sequence.
Example 10
Selection of Tooth-Binding Peptides Using Biopanning
[0242] The purpose of this prophetic Example is to describe how to
identify phage peptides that bind to teeth with high affinity.
[0243] Extracted human teeth, which may be obtained from a Dental
Office, are cleaned by brushing with soap solution, rinsed with
deionized water, and allowed to air-dry at room temperature. The
teeth are placed in a 15 mL centrifuge tube (Corning Inc., Acton,
Mass.), one tooth per tube. The teeth samples are treated for 1 h
with blocking buffer consisting of 1 mg/mL BSA in TBST-0.5%, and
then washed with TBST-0.5%. The teeth samples are incubated with
the phage library (Ph.D-12 Phage Display Peptide Library Kit) and
washed 10 times using the same conditions described in Example 1.
After the acidic elution step, described in Example 1, the teeth
samples are washed three more times with the elution buffer and
then washed three times with TBST-0.5%. The acid-treated teeth,
which have acid resistant phage peptides still attached, are used
to directly infect E. coli ER2738 cells as described in Example 2.
The amplified and isolated phages are contacted with a fresh tooth
sample and the biopanning procedure is repeated two more times.
After the third round of biopanning, the acid-treated teeth are
used to directly infect E. coli ER2738 cells, and the cells are
cultured as described in Example 1. Single black plaques are
randomly picked for DNA isolation and sequence analysis. The single
plaque lysates are prepared following the manufacture's
instructions (New England Biolabs) and the single stranded phage
genomic DNA is purified using the QIAprep Spin M13 Kit (Qiagen,
Valencia, Calif.) and sequenced using -96 gill sequencing primer,
as described in Example 1.
[0244] The identified peptide sequences will have a binding
affinity for teeth. The binding specificity and affinity of the
identified tooth-binding peptides is determined as described in
Example 6.
Examples 11-16
Hair Coloring Using Triblock Peptide-Based Body Surface Coloring
Reagents
[0245] The purpose of these Examples was to demonstrate the
coloring of hair using triblock peptide-based body surface coloring
reagents in combination with a carbon black pigment. The triblock
peptide-based body surface coloring reagents used consisted of an
empirically generated hair-binding peptide, a proline spacer, and a
carbon black-binding peptide.
[0246] The sequences of the triblock peptide-based body surface
coloring reagents used in these Examples are given in Table 14.
These peptide-based reagents were obtained from SynPep (Dublin,
Calif.). TABLE-US-00015 TABLE 14 Sequences of Triblock
Peptide-Based Body Surface Coloring Reagents Example Peptide
Sequence SEQ ID NO: 11 FHENWPS (carbon black- 138 binding peptide)
- PPP (spacer) - KKKK (hair- binding peptide) 12 FHENWPS (carbon
black- 139 binding peptide) - PPP (spacer) - HHHH (hair- binding
peptide) 13 FHENWPS (carbon black- 140 binding peptide) - PPP
(spacer) - RRRR (hair- binding peptide) 14 WHLSWSPVPLPT (carbon 141
black-binding peptide) - PPP (spacer) - KKKK (hair-binding peptide)
15 WHLSWSPVPLPT (carbon 142 black-binding peptide) - PPP (spacer) -
HHHH (hair-binding peptide) 16 WHLSWSPVPLPT (carbon 143
black-binding peptide) - PPP (spacer) - RRRR (hair-binding
peptide)
[0247] A 3 wt % solution of the peptide-based body surface coloring
reagent to be tested was prepared by dissolving the appropriate
amount of the peptide in water. To this aqueous peptide solution
was added 1.5 mL of a self-dispersed carbon black pigment
dispersion containing 14 wt % solids, prepared as described by Yeh
et al. in U.S. Pat. No. 6,852,156, Example 1. The resulting mixture
was stirred for 16 h.
[0248] A natural white hair swatch, obtained from International
Hair Importers, was immersed in the mixture with agitation for 30
min. After this time, the hair swatch was removed from the mixture,
allowed to air dry, and then was rinsed with water to remove the
unbound pigment. As a control, hair was colored using the same
procedure using the carbon black pigment without the peptide
reagent. For all the peptide-based body surface coloring reagents
tested, the color of the hair was significantly darker black after
the water rinse than the control hair sample that was colored
without the peptide-based body surface coloring reagent.
Examples 17-20
Biological Production of Triblock Peptide-Based Body Surface
Coloring Reagents
[0249] The purpose of these Examples was to prepare peptide-based
body surface coloring reagents using recombinant DNA and molecular
cloning techniques. The peptide-based body surface coloring
reagents were triblock structures comprised of hair-binding peptide
sequences, and carbon black-binding peptide sequences, separated by
peptide spacers. The peptides were expressed in E. coli as
inclusion bodies. Additional amino acid sequences (i.e., peptide
tags) were fused to the peptide-based body surface coloring reagent
sequences in order to promote inclusion body formation.
Construction of Production Strains
[0250] The sequences of the peptide-based body surface coloring
reagents are given in Table 15. DNA sequences were designed to
encode these peptides using favorable codons for E. coli and
avoiding sequence repeats and mRNA secondary structure. The gene
DNA sequence was designed by DNA 2.0, Inc. (Menlo Park, Calif.)
using proprietary software which is described by Gustafsson et al.
(Trends in Biotechnol. 22(7):346-355 (2004)). In each case the
sequence encoding the amino acid sequence was followed by two
termination codons and a recognition site for endonuclease AscI.
The GS amino acid sequence at the N-terminus was encoded by a
recognition site for endonuclease BamHI (GGA/TCC). The DNA
sequences are given by SEQ ID NOs:148-151. TABLE-US-00016 TABLE 15
Sequences of Triblock Peptide-Based Body Surface Coloring Reagents
Peptide DNA SEQ ID SEQ Example Peptide Sequence NO: ID NO: 17 DPG
(spacer) - WHLSWSPVPLPT (carbon 144 148 black-binding peptide) -
GGAGAGG (spacer) - WHLSWSPVPLPT (carbon black-binding peptide) -
AGGTSTSKASTT TTSSKTTTTSSKTTTTTSKTSTTSSSSTGGA (spacer) -
HEHKNQKETHQRHAA (hair- binding peptide) - GQGGYGGLGSQGAGRGGL GGQG
(spacer) - HEHKNQKETHQRHAA (hair-binding peptide) - GGKK (spacer)
18 GSDPG (spacer) - WHLSWSPVPLPT (carbon 145 149 black-binding
peptide) - GGAGGAG (spacer) - WHLSWSPVPLPT (carbon black-binding
peptide) - GGTSTSKASTTT TSSKTTTTSSKTTTTTSKTSTTSSSSTGG (spacer) -
NTSQLST (hair-binding peptide) - GSGGQGG (spacer) - NTSQLST
(hair-binding peptide) - GGPKK (spacer) 19 GSDPG (spacer) -
TPPELLHGAPRS (hair- 146 150 binding peptide) - GGAGGAG (spacer) -
WHLSWSPVPLPT (carbon black-binding peptide) - GK (spacer) 20 GSDPG
(spacer) - TPPELLHGAPRS (hair- 147 151 binding peptide) - GGAGGAG
(spacer) - TPPELLHGAPRS (hair-binding peptide) - GGAGGAV (spacer) -
WHLSWSPVPLPT (carbon black-binding peptide) - GGAGGAG (spacer) -
WHLSWSPVPLPT (carbon black-binding peptide) - GK (spacer)
[0251] Genes were assembled from synthetic oligonucleotides and
cloned into a standard plasmid cloning vector by DNA 2.0, Inc.
Sequences were verified by DNA sequencing by DNA 2.0, Inc.
[0252] The synthetic genes were excised from the cloning vector
with the endonuclease restriction enzymes BamHI and AscI and
ligated into an expression vector using standard recombinant DNA
methods. The vector pKSIC4-HC77623 was derived from the
commercially available vector pDEST17 (Invitrogen, Carlsbad,
Calif.). It includes sequences derived from the commercially
available vector pET31b (Novagen, Madison, Wis.) that encode a
fragment of the enzyme ketosteroid isomerase (KSI). The KSI
fragment was included as a fusion partner to promote partition of
the peptides into insoluble inclusion bodies in E. coli. The
KSI-encoding sequence from pET31b was modified using standard
mutagenesis procedures (QuickChange II, Stratagene, La Jolla,
Calif.) to include three additional Cys codons, in addition to the
one Cys codon found in the wild type KSI sequence. The plasmid
pKSIC4-HC77623, given by SEQ ID NO:152 and shown in FIG. 1, was
constructed using standard recombinant DNA methods, which are well
known to those skilled in the art.
[0253] The DNA sequences encoding the peptide-based body surface
coloring reagents (Table 15) were inserted into pKSIC4-HC77623 by
substituting for sequences in the vector between the BamHI and AscI
sites. Plasmid DNA containing the peptide encoding sequences and
vector DNA were digested with endonuclease restriction enzymes
BamHI and AscI, then the peptide-encoding sequences and vector DNA
were mixed and ligated by phage T4 DNA ligase using standard DNA
cloning procedures, which are well known to those skilled in the
art. Correct constructs, in which the sequences encoding the
peptide-based body surface coloring reagents were respectively
inserted into pKSIC4-HC77623, were identified by restriction
analysis and verified by DNA sequencing, using standard
methods.
[0254] In these constructs, the sequences encoding the peptides of
interest were substituted for those encoding HC77623. These
sequences were operably linked to the bacteriophage T7 gene 10
promoter and expressed as a fusion protein, fused with the variant
KSI partner.
[0255] To test the expression of the peptide-based reagents, the
expression plasmids were transformed into the BL21-AI E. coli
strain (Invitrogen, catalog no. C6070-03). To produce the
recombinant fusion peptides, 50 mL of LB-ampicillin broth (10 g/L
bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl, 100 mg/L
ampicillin, pH 7.0) was inoculated with the transformed bacteria
and the culture was shaken at 37.degree. C. until the OD.sub.600
reached 0.6. The expression was induced by adding 0.5 mL of 20 wt %
L-arabinose to the culture and shaking was continued for another 4
h. Analysis of the cell protein by polyacrylamide gel
electrophoresis demonstrated the production of the fusion
peptides.
Fermentation:
[0256] The recombinant E. coli strains, described above, were grown
in a 6-L fermentation, which was run in batch mode initially, and
then in fed-batch mode. The composition of the fermentation medium
is given in Table 16. The pH of the fermentation medium was 6.7.
The fermentation medium was sterilized by autoclaving, after which
the following sterilized components were added: thiamine
hydrochloride (4.5 mg/L), glucose (22.1 g/L), trace elements, see
Table 17 (10 mL/L), ampicillin (100 mg/L), and inoculum (seed) (125
mL). The pH was adjusted as needed using ammonium hydroxide (20 vol
%) or phosphoric acid (20 vol %). The added components were
sterilized either by autoclaving or filtration. TABLE-US-00017
TABLE 16 Composition of Fermentation Medium Component Concentration
KH.sub.2PO.sub.4 9 g/L (NH.sub.4).sub.2HPO.sub.4 4 g/L
MgSO.sub.4.cndot.7H.sub.2O 1.2 g/L Citric Acid 1.7 g/L Yeast
extract 5.0 g/L Mazu DF 204 Antifoam 0.1 mL/L
[0257] TABLE-US-00018 TABLE 17 Trace Elements Component
Concentration, mg/L EDTA 840 CoCl.sub.2.cndot.H.sub.2O 250
MnCl.sub.2.cndot.4H.sub.2O 1500 CuCl.sub.2.cndot.2H.sub.2O 150
H.sub.3BO.sub.3 300 Na.sub.2MoO.sub.4.cndot.2H.sub.2O 250
Zn(CH.sub.3COO).sub.2.cndot.H.sub.2O 1300 Ferric citrate 10000
[0258] The operating conditions for the fermentations are
summarized in Table 18. The initial concentration of glucose was
22.1 g/L. When the initial residual glucose was depleted, a
pre-scheduled, exponential glucose feed was initiated starting the
fed-batch phase of the fermentation run. The glucose feed (see
Tables 19 and 20) contained 500 g/L of glucose and was supplemented
with 5 g/L of yeast extract. The components of the feed medium were
sterilized either by autoclaving or filtration. The goal was to
sustain a specific growth rate of 0.13 h.sup.-1, assuming a yield
coefficient (biomass to glucose) of 0.25 g/g, and to maintain the
acetic acid levels in the fermentation vessel at very low values
(i.e., less than 0.2 g/L). The glucose feed continued until the end
of the run. Induction was initiated with a bolus of 2 g/L of
L-arabinose at the selected time (i.e., 15 h of elapsed
fermentation time). A bolus to deliver 5 g of yeast extract per
liter of fermentation broth was added to the fermentation vessel at
the following times: 1 h prior to induction, at induction time, and
1 h after induction time. The fermentation run was terminated after
19.97 h of elapsed fermentation time, and 4.97 h after the
induction time. TABLE-US-00019 TABLE 18 Fermentation Operating
Conditions Condition Initial Minimum Maximum Stirring 220 rpm 220
rpm 1200 rpm Air Flow 3 SLPM 3 SLPM 30 SLPM Temperature 37.degree.
C. 37.degree. C. 37.degree. C. pH 6.7 6.7 6.7 Pressure 0.500 atm
0.500 atm 0.500 atm (50.7 kPa) (50.7 kPa) (50.7 kPa) Dissolved
O.sub.2* 20% 20% 20% *Cascade stirrer, then air flow.
[0259] TABLE-US-00020 TABLE 19 Composition of Feed Medium Component
Concentration MgSO.sub.4.cndot.7H.sub.2O 2.0 g/L Glucose 500 g/L
Ampicillin 150 mg/L (NH.sub.4).sub.2HPO.sub.4 4 g/L
KH.sub.2PO.sub.4 9 g/L Yeast extract 5.0 g/L Trace Elements - Feed
(Table 5) 10 mL/L
[0260] TABLE-US-00021 TABLE 20 Trace Elements - Feed Component
Concentration, mg/L EDTA 1300 CoCl.sub.2.cndot.H.sub.2O 400
MnCl.sub.2.cndot.4H.sub.2O 2350 CuCl.sub.2.cndot.2H.sub.2O 250
H.sub.3BO.sub.3 500 Na.sub.2MoO.sub.4.cndot.2H.sub.2O 400
Zn(CH.sub.3COO).sub.2.cndot.H.sub.2O 1600 Ferric citrate 4000
Isolation and Purification of Peptides:
[0261] After completion of the fermentation run, the entire
fermentation broth was passed three times through an APV model 2000
Gaulin type homogenizer at 12,000 psi (82,700 kPa). The broth was
cooled to below 5.degree. C. prior to each homogenization. The
homogenized broth was immediately processed through a Westfalia
WhisperFuge.TM. (Westfalia Separator Inc., Northvale, N.J.) stacked
disc centrifuge at 600 mL/min and 12,000 RCF to separate inclusion
bodies from suspended cell debris and dissolved impurities. The
recovered paste was re-suspended at 15 g/L (dry basis) in water and
the pH was adjusted to a value between 8.0 and 10.0 using NaOH. The
pH was chosen to help remove cell debris from the inclusion bodies
without dissolving the inclusion body proteins. The suspension was
passed through the APV 2000 Gaulin type homogenizer at 12,000 psi
(82,700 kPa) for a single pass to provide rigorous mixing. The
homogenized high pH suspension was immediately processed in a
Westfalia WhisperFuge.TM. stacked disc centrifuge at 600 mL/min and
12,000 RCF to separate the washed inclusion bodies from suspended
cell debris and dissolved impurities. The recovered paste was
resuspended at 15 gm/L (dry basis) in pure water. The suspension
was passed through the APV 2000 Gaulin type homogenizer at 12,000
psi (82,700 kPa) for a single pass to provide rigorous washing. The
homogenized suspension was immediately processed in a Westfalia
WhisperFuge.TM. stacked disc centrifuge at 600 mL/min and 12,000
RCF to separate the washed inclusion bodies from residual suspended
cell debris and NaOH.
[0262] The recovered paste was resuspended in pure water at 25 g/L
(dry basis) and the pH of the mixture was adjusted to 2.2 using
HCl. If the peptide being recovered contained cysteine residues,
dithiothreitol (DTT, 10 mM) was added to break disulfide bonds. The
acidified suspension was heated to 70.degree. C. for 5 to 14 h to
complete cleavage of the DP site separating the fusion peptide from
the product peptide without damaging the target peptide. The
product slurry was adjusted to pH 5.1 (note: the pH used here may
vary depending on the solubility of the peptide being recovered)
using NaOH and then was cooled to 5.degree. C. and held for 12 h.
The mixture was centrifuged at 9000 RCF for 30 min and the
supernatant was decanted. The supernatant was then filtered with a
0.2 .mu.m membrane. For some low solubility peptides, multiple
washes of the pellet were required to increase peptide
recovery.
[0263] The filtered product was pH adjusted to 2.0 and mixed with
sufficient acetonitrile to yield a solution that was 10 vol %
acetonitrile in order to stabilize the samples. This solution was
loaded in a 22.times.250 mm or a 50.times.250 mm reverse phase
chromatography column containing 10 to 15 .mu.m C18 media which was
preconditioned with 10 vol % acetonitrile, 90 vol % water with 0.1
vol % trifluoroacetic acid (TFA). The product was recovered in a
purified state by eluting the column with a gradient of water and
acetonitrile, ramping from 10 vol % to 40 vol % acetonitrile in
water with TFA at 0.1 vol %. The eluent containing the product
peptide was collected and concentrated by vacuum evaporation by a
factor of 2:1 before lyophilization. Spectrophotometric detection
at 220 and 278 nm was used to monitor and track elution of the
product peptide.
Example 21
Hair Coloring Using a Triblock Peptide-Based Body Surface Coloring
Reagent
[0264] The purpose of this Example was to demonstrate the coloring
of hair using a triblock peptide-based body surface coloring
reagent in combination with a carbon black pigment. The color
retention was quantified using a spectrophotometric measurement
technique.
[0265] A self-dispersed carbon black pigment dispersion containing
14 wt % solids, prepared as described by Yeh et al. in U.S. Pat.
No. 6,852,156, Example 1, was diluted 1:10 with water. Twenty-five
milligrams of the peptide-based body surface coloring reagent given
as SEQ ID NO:147, (Example 20) was dissolved in 5 g of water. Then,
10 g of the diluted carbon black pigment dispersion was slowly
added to the peptide solution and the solution was mixed for at
least 60 min.
[0266] A natural white hair swatch, obtained from International
Hair Importers, was immersed in the mixture with agitation for 30
min. After this time, the hair swatch was removed from the mixture,
allowed to air dry, and then was rinsed with water to remove the
unbound pigment. As a control, hair was colored using the same
procedure using the carbon black pigment without the peptide
reagent.
[0267] The color intensity after the water rinse was measured using
a X-Rite.RTM. SP78.TM. Sphere Spectrophotometer (X-Rite, Inc.,
Grandville, Mich.), by placing the colored hair sample into the
photosensor and calculating L*, a* and b* parameters representing
the photometer response. An initial baseline L* value was measured
for the uncolored hair and all measurements were the average of
five individual determinations. Delta E values were calculated
using equation 1 below: Delta
E=((L*.sub.1-L*.sub.2).sup.2+(a.sub.1-a.sub.2).sup.2+(b.sub.1-b.sub-
.2).sup.2).sup.1/2 (1)
[0268] where L*=the lightness variable and a* and b* are the
chromaticity coordinates of CIELAB colorspace as defined by the
International Commission of Illumination (CIE) (Minolta, Precise
Color Communication--Color Control From Feeling to Instrumentation,
Minolta Camera Co., 1996). Larger Delta E value are indicative of
better color retention. The results are summarized in Table 21.
TABLE-US-00022 TABLE 21 Results of Color Retention After Water
Rinse Sample Delta E Peptide-based body surface 31.4 coloring
reagent plus pigment Control, pigment alone 18.2
[0269] As can be seen from the data in Table 21, the color
retention after the water rinse was significantly higher for the
sample treated with the peptide-based body surface coloring reagent
and the pigment than with the control sample, which was treated
with only the pigment.
Sequence CWU 1
1
156 1 8 PRT Artificial Sequence Hair-binding peptide 1 Leu Glu Ser
Thr Pro Lys Met Lys 1 5 2 7 PRT Artificial Sequence Skin-binding
peptide 2 Phe Thr Gln Ser Leu Pro Arg 1 5 3 12 PRT Artificial
Sequence Hair-binding peptide 3 Ser Val Ser Val Gly Met Lys Pro Ser
Pro Arg Pro 1 5 10 4 12 PRT Artificial Sequence Hair-binding
peptide 4 Leu Asp Val Glu Ser Tyr Lys Gly Thr Ser Met Pro 1 5 10 5
12 PRT Artificial Sequence Hair-binding peptide. 5 Arg Val Pro Asn
Lys Thr Val Thr Val Asp Gly Ala 1 5 10 6 12 PRT Artificial Sequence
Hair-binding peptide 6 Asp Arg His Lys Ser Lys Tyr Ser Ser Thr Lys
Ser 1 5 10 7 12 PRT Artificial Sequence Hair-binding peptide 7 Lys
Asn Phe Pro Gln Gln Lys Glu Phe Pro Leu Ser 1 5 10 8 12 PRT
Artificial Sequence Hair-binding peptide 8 Gln Arg Asn Ser Pro Pro
Ala Met Ser Arg Arg Asp 1 5 10 9 12 PRT Artificial Sequence
Hair-binding peptide 9 Thr Arg Lys Pro Asn Met Pro His Gly Gln Tyr
Leu 1 5 10 10 12 PRT Artificial Sequence Hair-binding peptide 10
Lys Pro Pro His Leu Ala Lys Leu Pro Phe Thr Thr 1 5 10 11 12 PRT
Artificial Sequence Hair-binding peptide 11 Asn Lys Arg Pro Pro Thr
Ser His Arg Ile His Ala 1 5 10 12 12 PRT Artificial Sequence
Hair-binding peptide 12 Asn Leu Pro Arg Tyr Gln Pro Pro Cys Lys Pro
Leu 1 5 10 13 12 PRT Artificial Sequence Hair-binding peptide 13
Arg Pro Pro Trp Lys Lys Pro Ile Pro Pro Ser Glu 1 5 10 14 12 PRT
Artificial Sequence Hair-binding peptide 14 Arg Gln Arg Pro Lys Asp
His Phe Phe Ser Arg Pro 1 5 10 15 12 PRT Artificial Sequence
Hair-binding peptide MISC_FEATURE (6)..(6) Xaa = Thr or Pro
MISC_FEATURE (12)..(12) Xaa = Glu or Ala 15 Ser Val Pro Asn Lys Xaa
Val Thr Val Asp Gly Xaa 1 5 10 16 12 PRT Artificial Sequence
Hair-binding peptide 16 Thr Thr Lys Trp Arg His Arg Ala Pro Val Ser
Pro 1 5 10 17 12 PRT Artificial Sequence Hair-binding peptide 17
Trp Leu Gly Lys Asn Arg Ile Lys Pro Arg Ala Ser 1 5 10 18 12 PRT
Artificial Sequence Hair-binding peptide 18 Ser Asn Phe Lys Thr Pro
Leu Pro Leu Thr Gln Ser 1 5 10 19 12 PRT Artificial Sequence
Hair-binding peptide 19 Lys Glu Leu Gln Thr Arg Asn Val Val Gln Arg
Glu 1 5 10 20 12 PRT Artificial Sequence Hair-binding peptide 20
Thr Pro Thr Ala Asn Gln Phe Thr Gln Ser Val Pro 1 5 10 21 12 PRT
Artificial Sequence Hair-binding peptide 21 Ala Ala Gly Leu Ser Gln
Lys His Glu Arg Asn Arg 1 5 10 22 12 PRT Artificial Sequence
Hair-binding peptide 22 Glu Thr Val His Gln Thr Pro Leu Ser Asp Arg
Pro 1 5 10 23 12 PRT Artificial Sequence Hair-binding peptide 23
Leu Pro Ala Leu His Ile Gln Arg His Pro Arg Met 1 5 10 24 12 PRT
Artificial Sequence Hair-binding peptide 24 Gln Pro Ser His Ser Gln
Ser His Asn Leu Arg Ser 1 5 10 25 12 PRT Artificial Sequence
Hair-binding peptide 25 Arg Gly Ser Gln Lys Ser Lys Pro Pro Arg Pro
Pro 1 5 10 26 12 PRT Artificial Sequence Hair-binding peptide 26
Thr His Thr Gln Lys Thr Pro Leu Leu Tyr Tyr His 1 5 10 27 12 PRT
Artificial Sequence Hair-binding peptide 27 Thr Lys Gly Ser Ser Gln
Ala Ile Leu Lys Ser Thr 1 5 10 28 7 PRT Artificial Sequence
Hair-binding peptide 28 Asp Leu His Thr Val Tyr His 1 5 29 7 PRT
Artificial Sequence Hair-binding peptide 29 His Ile Lys Pro Pro Thr
Arg 1 5 30 7 PRT Artificial Sequence Hair-binding peptide 30 His
Pro Val Trp Pro Ala Ile 1 5 31 7 PRT Artificial Sequence
Hair-binding peptide 31 Met Pro Leu Tyr Tyr Leu Gln 1 5 32 26 PRT
Artificial Sequence Hair-binding peptide 32 His Leu Thr Val Pro Trp
Arg Gly Gly Gly Ser Ala Val Pro Phe Tyr 1 5 10 15 Ser His Ser Gln
Ile Thr Leu Pro Asn His 20 25 33 41 PRT Artificial Sequence
Hair-binding peptide 33 Gly Pro His Asp Thr Ser Ser Gly Gly Val Arg
Pro Asn Leu His His 1 5 10 15 Thr Ser Lys Lys Glu Lys Arg Glu Asn
Arg Lys Val Pro Phe Tyr Ser 20 25 30 His Ser Val Thr Ser Arg Gly
Asn Val 35 40 34 7 PRT Artificial Sequence Hair-binding peptide 34
Lys His Pro Thr Tyr Arg Gln 1 5 35 7 PRT Artificial Sequence
Hair-binding peptide 35 His Pro Met Ser Ala Pro Arg 1 5 36 7 PRT
Artificial Sequence Hair-binding peptide 36 Met Pro Lys Tyr Tyr Leu
Gln 1 5 37 7 PRT Artificial Sequence Hair-binding peptide 37 Met
His Ala His Ser Ile Ala 1 5 38 7 PRT Artificial Sequence
Hair-binding peptide 38 Thr Ala Ala Thr Thr Ser Pro 1 5 39 7 PRT
Artificial Sequence Hair-binding peptide 39 Leu Gly Ile Pro Gln Asn
Leu 1 5 40 12 PRT Artificial Sequence Hair-binding peptide 40 Ala
Lys Pro Ile Ser Gln His Leu Gln Arg Gly Ser 1 5 10 41 12 PRT
Artificial Sequence Hair-binding peptide 41 Ala Pro Pro Thr Pro Ala
Ala Ala Ser Ala Thr Thr 1 5 10 42 12 PRT Artificial Sequence
Hair-binding peptide 42 Asp Pro Thr Glu Gly Ala Arg Arg Thr Ile Met
Thr 1 5 10 43 12 PRT Artificial Sequence Hair-binding peptide 43
Glu Gln Ile Ser Gly Ser Leu Val Ala Ala Pro Trp 1 5 10 44 12 PRT
Artificial Sequence Hair-binding peptide 44 Leu Asp Thr Ser Phe Pro
Pro Val Pro Phe His Ala 1 5 10 45 11 PRT Artificial Sequence
Hair-binding peptide 45 Leu Pro Arg Ile Ala Asn Thr Trp Ser Pro Ser
1 5 10 46 12 PRT Artificial Sequence Hair-binding peptide 46 Arg
Thr Asn Ala Ala Asp His Pro Ala Ala Val Thr 1 5 10 47 12 PRT
Artificial Sequence Hair-binding peptide 47 Ser Leu Asn Trp Val Thr
Ile Pro Gly Pro Lys Ile 1 5 10 48 12 PRT Artificial Sequence
Hair-binding peptide 48 Thr Asp Met Gln Ala Pro Thr Lys Ser Tyr Ser
Asn 1 5 10 49 12 PRT Artificial Sequence Hair-binding peptide 49
Thr Ile Met Thr Lys Ser Pro Ser Leu Ser Cys Gly 1 5 10 50 12 PRT
Artificial Sequence Hair-binding peptide 50 Thr Pro Ala Leu Asp Gly
Leu Arg Gln Pro Leu Arg 1 5 10 51 12 PRT Artificial Sequence
Hair-binding peptide 51 Thr Tyr Pro Ala Ser Arg Leu Pro Leu Leu Ala
Pro 1 5 10 52 12 PRT Artificial Sequence Hair-binding peptide 52
Ala Lys Thr His Lys His Pro Ala Pro Ser Tyr Ser 1 5 10 53 12 PRT
Artificial Sequence Hair-binding and nail-binding peptide 53 Tyr
Pro Ser Phe Ser Pro Thr Tyr Arg Pro Ala Phe 1 5 10 54 12 PRT
Artificial Sequence Hair-binding peptide 54 Thr Asp Pro Thr Pro Phe
Ser Ile Ser Pro Glu Arg 1 5 10 55 20 PRT Artificial Sequence
Hair-binding peptide 55 Cys Ala Ala Gly Cys Cys Thr Cys Ala Gly Cys
Gly Ala Cys Cys Gly 1 5 10 15 Ala Ala Thr Ala 20 56 12 PRT
Artificial Sequence Hair-binding peptide 56 Trp His Asp Lys Pro Gln
Asn Ser Ser Lys Ser Thr 1 5 10 57 12 PRT Artificial Sequence
Hair-binding peptide 57 Asn Glu Val Pro Ala Arg Asn Ala Pro Trp Leu
Val 1 5 10 58 13 PRT Artificial Sequence Hair-binding peptide 58
Asn Ser Pro Gly Tyr Gln Ala Asp Ser Val Ala Ile Gly 1 5 10 59 12
PRT Artificial Sequence Hair-binding peptide 59 Thr Gln Asp Ser Ala
Gln Lys Ser Pro Ser Pro Leu 1 5 10 60 12 PRT Artificial Sequence
Nail-binding peptide 60 Ala Leu Pro Arg Ile Ala Asn Thr Trp Ser Pro
Ser 1 5 10 61 12 PRT Artificial Sequence Skin-binding peptide 61
Thr Pro Phe His Ser Pro Glu Asn Ala Pro Gly Ser 1 5 10 62 20 DNA
Artificial Sequence Primer 62 ccctcatagt tagcgtaacg 20 63 12 PRT
Artificial Sequence Control peptide 63 Lys His Gly Pro Asp Leu Leu
Arg Ser Ala Pro Arg 1 5 10 64 16 PRT Artificial Sequence
Cysteine-attached hair-binding peptide 64 Arg Thr Asn Ala Ala Asp
His Pro Ala Ala Val Thr Gly Gly Gly Cys 1 5 10 15 65 8 PRT
Artificial Sequence Caspase 3 cleavage site 65 Leu Glu Ser Gly Asp
Glu Val Asp 1 5 66 12 PRT Artificial Sequence Hair-binding peptide
66 Thr Pro Pro Glu Leu Leu His Gly Asp Pro Arg Ser 1 5 10 67 20 DNA
Artificial Sequence Primer 67 caagcctcag cgaccgaata 20 68 23 DNA
Artificial Sequence Primer 68 cgtaacactg agtttcgtca cca 23 69 12
PRT Artificial Sequence Hair-binding peptide 69 Thr Pro Pro Thr Asn
Val Leu Met Leu Ala Thr Lys 1 5 10 70 7 PRT Artificial Sequence
Hair-binding peptide 70 Asn Thr Ser Gln Leu Ser Thr 1 5 71 14 PRT
Artificial Sequence Biotinylated hair-binding peptide MISC_FEATURE
(13)..(13) Biotinylated MISC_FEATURE (14)..(14) Amidated 71 Arg Thr
Asn Ala Ala Asp His Pro Ala Ala Val Thr Lys Cys 1 5 10 72 14 PRT
Artificial Sequence Biotinylated hair-binding peptide MISC_FEATURE
(13)..(13) Biotinylated MISC_FEATURE (14)..(14) Amidated 72 Ala Leu
Pro Arg Ile Ala Asn Thr Trp Ser Pro Ser Lys Cys 1 5 10 73 14 PRT
Artificial Sequence Biotinylated hair-binding peptide MISC_FEATURE
(13)..(13) Biotinylated MISC_FEATURE (14)..(14) Amidated 73 Thr Pro
Pro Glu Leu Leu His Gly Asp Pro Arg Ser Lys Cys 1 5 10 74 14 PRT
Artificial Sequence Biotinylated skin-binding peptide MISC_FEATURE
(13)..(13) Biotinylated MISC_FEATURE (14)..(14) Amidated 74 Thr Pro
Phe His Ser Pro Glu Asn Ala Pro Gly Ser Lys Cys 1 5 10 75 15 PRT
Artificial Sequence Hair conditioner resistant hair-binding peptide
MISC_FEATURE (1)..(1) Fluoroenylmethoxlcarbonyl (Fmoc)-protected
MISC_FEATURE (1)..(1) 2,2,6,4,7-pentamethyldihydrobenzofuran-
5sulfonyl-protected MISC_FEATURE (2)..(2) t-butyl-protected
MISC_FEATURE (3)..(3) Trityl-protected MISC_FEATURE (6)..(6)
t-butoxyl-protected MISC_FEATURE (7)..(7) Trityl-protected
MISC_FEATURE (12)..(12) t-butyl-protected 75 Ser Thr Leu His Lys
Tyr Lys Ser Gln Asp Pro Thr Pro His His 1 5 10 15 76 7 PRT
Artificial Sequence Hair-binding peptide 76 Asn Thr Pro Lys Glu Asn
Trp 1 5 77 7 PRT Artificial Sequence Hair-binding peptide 77 Asn
Thr Pro Ala Ser Asn Arg 1 5 78 7 PRT Artificial Sequence
Hair-binding peptide 78 Pro Arg Gly Met Leu Ser Thr 1 5 79 7 PRT
Artificial Sequence Hair-binding peptide 79 Pro Pro Thr Tyr Leu Ser
Thr 1 5 80 12 PRT Artificial Sequence Hair-binding peptide 80 Thr
Ile Pro Thr His Arg Gln His Asp Tyr Arg Ser 1 5 10 81 7 PRT
Artificial Sequence Hair-binding peptide 81 Thr Pro Pro Thr His Arg
Leu 1 5 82 7 PRT Artificial Sequence Hair-binding peptide 82 Leu
Pro Thr Met Ser Thr Pro 1 5 83 7 PRT Artificial Sequence
Hair-binding peptide 83 Leu Gly Thr Asn Ser Thr Pro 1 5 84 12 PRT
Artificial Sequence Hair-binding peptide 84 Thr Pro Leu Thr Gly Ser
Thr Asn Leu Leu Ser Ser 1 5 10 85 7 PRT Artificial Sequence
Hair-binding peptide 85 Thr Pro Leu Thr Lys Glu Thr 1 5 86 7 PRT
Artificial Sequence Hair-binding peptide 86 Gln Gln Ser His Asn Pro
Pro 1 5 87 7 PRT Artificial Sequence Hair-binding peptide 87 Thr
Gln Pro His Asn Pro Pro 1 5 88 12 PRT Artificial Sequence
Hair-binding peptide 88 Ser Thr Asn Leu Leu Arg Thr Ser Thr Val His
Pro 1 5 10 89 12 PRT Artificial Sequence Hair-binding peptide 89
His Thr Gln Pro Ser Tyr Ser Ser Thr Asn Leu Phe 1 5 10 90 7 PRT
Artificial Sequence Hair-binding peptide 90 Ser Leu Leu Ser Ser His
Ala 1 5 91 12 PRT Artificial Sequence Hair-binding peptide 91 Gln
Gln Ser Ser Ile Ser Leu Ser Ser His Ala Val 1 5 10 92 7 PRT
Artificial Sequence Hair-binding peptide 92 Asn Ala Ser Pro Ser Ser
Leu 1 5 93 7 PRT Artificial Sequence Hair-binding peptide 93 His
Ser Pro Ser Ser Leu Arg 1 5 94 7 PRT Artificial Sequence
Hair-binding peptide MISC_FEATURE (2)..(2) Xaa = His, Arg, or Asn
94 Lys Xaa Ser His His Thr His 1 5 95 7 PRT Artificial Sequence
Hair-binding peptide MISC_FEATURE (2)..(2) Xaa = His, Arg, or Asn
95 Glu Xaa Ser His His Thr His 1 5 96 7 PRT Artificial Sequence
Hair-binding peptide 96 Leu Glu Ser Thr Ser Leu Leu 1 5 97 7 PRT
Artificial Sequence Hair-binding peptide 97 Thr Pro Leu Thr Lys Glu
Thr 1 5 98 7 PRT Artificial Sequence Hair-binding peptide 98 Lys
Gln Ser His Asn Pro Pro 1 5 99 12 PRT Artificial Sequence
Skin-binding sequence 99 Lys Gln Ala Thr Phe Pro Pro Asn Pro Thr
Ala Tyr 1 5 10 100 12 PRT Artificial Sequence Skin-binding peptide
100 His Gly His Met Val Ser Thr Ser Gln Leu Ser Ile 1 5 10 101 7
PRT Artificial Sequence Skin-binding peptide 101 Leu Ser Pro Ser
Arg Met Lys 1 5 102 7 PRT Artificial Sequence Skin-binding peptide
102 Leu Pro Ile Pro Arg Met Lys 1 5 103 7 PRT Artificial Sequence
Skin-binding peptide 103 His Gln Arg Pro Tyr Leu Thr 1 5 104 7 PRT
Artificial Sequence Skin-binding peptide 104 Phe Pro Pro Leu Leu
Arg Leu 1 5 105 12 PRT Artificial Sequence Empirically generated
hair and skin-binding peptide 105 Lys Arg Gly Arg His Lys Arg Pro
Lys Arg His Lys 1 5 10 106 7 PRT Artificial Sequence Empirically
generated hair and skin-binding peptide 106 Arg Leu Leu Arg Leu Leu
Arg 1 5 107 12 PRT Artificial Sequence Empirically generated hair
and skin-binding peptide 107 His Lys Pro Arg Gly Gly Arg Lys Lys
Ala Leu His 1 5 10 108 18 PRT Artificial Sequence Empirically
generated hair and skin-binding peptide 108 Lys Pro Arg Pro Pro His
Gly Lys Lys His Arg Pro Lys His Arg Pro 1 5 10 15 Lys Lys 109 18
PRT Artificial Sequence Empirically generated hair and skin-binding
peptide 109 Arg Gly Arg Pro Lys Lys Gly His Gly Lys Arg Pro Gly His
Arg Ala 1 5 10 15 Arg Lys 110 7 PRT Artificial Sequence
Pigment-binding peptide 110 Met Pro Pro Pro Leu Met Gln 1 5 111 7
PRT Artificial Sequence Pigment-binding peptide 111 Phe His Glu Asn
Trp Pro Ser 1 5 112 12 PRT Artificial Sequence Pigment-binding
peptide 112 Arg Thr Ala Pro Thr Thr Pro Leu Leu Leu Ser Leu 1 5 10
113 12 PRT Artificial Sequence Pigment-binding peptide 113 Trp His
Leu Ser Trp Ser Pro Val Pro Leu Pro Thr 1 5 10 114 7 PRT Artificial
Sequence Pigment-binding peptide 114 Pro His Ala Arg Leu Val Gly 1
5 115 14 PRT Artificial Sequence Pigment-binding peptide 115 Asn
Ile Pro Tyr His His Pro Asn Ile Pro Tyr His His Pro 1 5 10 116 7
PRT Artificial Sequence Pigment-binding peptide 116 Thr Thr Met Pro
Ala Ile Pro 1 5 117 7 PRT Artificial Sequence Pigment-binding
peptide 117 His Asn Leu Pro Pro Arg Ser 1 5 118 12 PRT Artificial
Sequence Pigment-binding peptide 118 Ala His Lys Thr Gln Met Gly
Val Arg Gln Pro Ala 1 5 10 119 12 PRT Artificial Sequence
Pigment-binding peptide 119 Ala Asp Asn Val Gln Met Gly Val Ser His
Thr Pro 1 5 10 120 12 PRT Artificial Sequence Pigment-binding
peptide 120 Ala His Asn Ala Gln Met Gly Val Ser His Pro Pro 1 5 10
121 12 PRT Artificial Sequence Pigment-binding peptide 121 Ala Asp
Tyr Val Gly Met Gly Val Ser His Arg Pro 1 5 10 122 12 PRT
Artificial Sequence Pigment-binding peptide 122 Ser Val Ser Val Gly
Met Lys Pro Ser Pro Arg Pro 1 5 10 123 7 PRT Artificial Sequence
Pigment-binding peptide 123 Tyr Pro Asn Thr Ala Leu Val 1 5 124 7
PRT Artificial Sequence Pigment-binding peptide 124 Val Ala Thr Arg
Ile Val Ser 1 5 125 12 PRT Artificial Sequence Pigment-binding
peptide 125 His Ser Leu Lys Asn Ser Met Leu Thr Val Met Ala 1 5 10
126 7 PRT Artificial Sequence Pigment-binding peptide 126 Asn Tyr
Pro Thr Gln Ala Pro 1 5 127 7 PRT Artificial Sequence
Pigment-binding peptide 127 Lys Cys Cys Tyr Ser Val Gly 1 5 128 12
PRT Artificial Sequence
Pigment-binding peptide 128 Arg His Asp Leu Asn Thr Trp Leu Pro Pro
Val Lys 1 5 10 129 12 PRT Pigment-binding peptide 129 Glu Ile Ser
Leu Pro Ala Lys Leu Pro Ser Ala Ser 1 5 10 130 12 PRT Artificial
Sequence Pigment-binding peptide 130 Tyr Val Cys Glu Gly Ile His
Pro Cys Pro Arg Pro 1 5 10 131 12 PRT Artificial Sequence
Pigment-binding peptide 131 Ser Asp Tyr Val Gly Met Arg Pro Ser Pro
Arg His 1 5 10 132 12 PRT Artificial Sequence Pigment-binding
peptide 132 Ser Asp Tyr Val Gly Met Arg Leu Ser Pro Ser Gln 1 5 10
133 12 PRT Artificial Sequence Pigment-binding peptide 133 Ser Val
Ser Val Gly Ile Gln Pro Ser Pro Arg Pro 1 5 10 134 12 PRT
Artificial Sequence Pigment-binding peptide 134 Tyr Val Ser Val Gly
Ile Lys Pro Ser Pro Arg Pro 1 5 10 135 37 PRT Artificial Sequence
Peptide spacer 135 Thr Ser Thr Ser Lys Ala Ser Thr Thr Thr Thr Ser
Ser Lys Thr Thr 1 5 10 15 Thr Thr Ser Ser Lys Thr Thr Thr Thr Thr
Ser Lys Thr Ser Thr Thr 20 25 30 Ser Ser Ser Ser Thr 35 136 22 PRT
Artificial Sequence Peptide spacer 136 Gly Gln Gly Gly Tyr Gly Gly
Leu Gly Ser Gln Gly Ala Gly Arg Gly 1 5 10 15 Gly Leu Gly Gly Gln
Gly 20 137 10 PRT Artificial Sequence Peptide spacer 137 Gly Pro
Gly Gly Tyr Gly Pro Gly Gln Gln 1 5 10 138 14 PRT Artificial
Sequence Triblock peptide-based body surface reagent 138 Phe His
Glu Asn Trp Pro Ser Pro Pro Pro Lys Lys Lys Lys 1 5 10 139 14 PRT
Artificial Sequence Triblock peptide-based body surface reagent 139
Phe His Glu Asn Trp Pro Ser Pro Pro Pro His His His His 1 5 10 140
14 PRT Artificial Sequence Triblock peptide-based body surface
reagent 140 Phe His Glu Asn Trp Pro Ser Pro Pro Pro Arg Arg Arg Arg
1 5 10 141 19 PRT Artificial Sequence Triblock peptide-based body
surface reagent 141 Trp His Leu Ser Trp Ser Pro Val Pro Leu Pro Thr
Pro Pro Pro Lys 1 5 10 15 Lys Lys Lys 142 19 PRT Artificial
Sequence Triblock peptide-based body surface reagent 142 Trp His
Leu Ser Trp Ser Pro Val Pro Leu Pro Thr Pro Pro Pro His 1 5 10 15
His His His 143 19 PRT Artificial Sequence Triblock peptide-based
body surface reagent 143 Trp His Leu Ser Trp Ser Pro Val Pro Leu
Pro Thr Pro Pro Pro Arg 1 5 10 15 Arg Arg Arg 144 133 PRT
Artificial Sequence Multicopy peptide-based body surface coloring
reagent 144 Asp Pro Gly Trp His Leu Ser Trp Ser Pro Val Pro Leu Pro
Thr Gly 1 5 10 15 Gly Ala Gly Ala Gly Gly Trp His Leu Ser Trp Ser
Pro Val Pro Leu 20 25 30 Pro Thr Ala Gly Gly Thr Ser Thr Ser Lys
Ala Ser Thr Thr Thr Thr 35 40 45 Ser Ser Lys Thr Thr Thr Thr Ser
Ser Lys Thr Thr Thr Thr Thr Ser 50 55 60 Lys Thr Ser Thr Thr Ser
Ser Ser Ser Thr Gly Gly Ala His Glu His 65 70 75 80 Lys Asn Gln Lys
Glu Thr His Gln Arg His Ala Ala Gly Gln Gly Gly 85 90 95 Tyr Gly
Gly Leu Gly Ser Gln Gly Ala Gly Arg Gly Gly Leu Gly Gly 100 105 110
Gln Gly His Glu His Lys Asn Gln Lys Glu Thr His Gln Arg His Ala 115
120 125 Ala Gly Gly Lys Lys 130 145 103 PRT Artificial Sequence
Multicopy peptide-based body surface coloring reagent 145 Gly Ser
Asp Pro Gly Trp His Leu Ser Trp Ser Pro Val Pro Leu Pro 1 5 10 15
Thr Gly Gly Ala Gly Gly Ala Gly Trp His Leu Ser Trp Ser Pro Val 20
25 30 Pro Leu Pro Thr Gly Gly Thr Ser Thr Ser Lys Ala Ser Thr Thr
Thr 35 40 45 Thr Ser Ser Lys Thr Thr Thr Thr Ser Ser Lys Thr Thr
Thr Thr Thr 50 55 60 Ser Lys Thr Ser Thr Thr Ser Ser Ser Ser Thr
Gly Gly Asn Thr Ser 65 70 75 80 Gln Leu Ser Thr Gly Ser Gly Gly Gln
Gly Gly Asn Thr Ser Gln Leu 85 90 95 Ser Thr Gly Gly Pro Lys Lys
100 146 38 PRT Artificial Sequence Peptide-based body surface
coloring reagent 146 Gly Ser Asp Pro Gly Thr Pro Pro Glu Leu Leu
His Gly Ala Pro Arg 1 5 10 15 Ser Gly Gly Ala Gly Gly Ala Gly Trp
His Leu Ser Trp Ser Pro Val 20 25 30 Pro Leu Pro Thr Gly Lys 35 147
76 PRT Artificial Sequence Multicopy peptide-based body surface
coloring reagent 147 Gly Ser Asp Pro Gly Thr Pro Pro Glu Leu Leu
His Gly Ala Pro Arg 1 5 10 15 Ser Gly Gly Ala Gly Gly Ala Gly Thr
Pro Pro Glu Leu Leu His Gly 20 25 30 Ala Pro Arg Ser Gly Gly Ala
Gly Gly Ala Val Trp His Leu Ser Trp 35 40 45 Ser Pro Val Pro Leu
Pro Thr Gly Gly Ala Gly Gly Ala Gly Trp His 50 55 60 Leu Ser Trp
Ser Pro Val Pro Leu Pro Thr Gly Lys 65 70 75 148 419 DNA Artificial
Sequence Coding sequence for peptide-based body surface coloring
reagent 148 ggatccgacc ctggttggca cctgtcttgg tcccctgttc ctctgccgac
cggtggtgca 60 ggcgctggtg gctggcacct gtcctggagc cctgtcccgc
tgccgactgc cggcggtact 120 tctacctcca aagcgtccac gaccactacc
agcagcaaaa ccacgaccac cagctctaaa 180 actaccacta ccaccagcaa
gacctccact accagctctt cttctactgg tggcgcccat 240 gaacataaaa
accagaaaga aacccaccag cgtcacgctg cgggtcaagg cggttacggt 300
ggcctgggta gccagggtgc aggtcgcggt ggtctgggtg gccagggtca cgaacacaag
360 aatcagaaag aaacgcacca gcgccacgct gcgggtggca aaaagtaata
aggcgcgcc 419 149 323 DNA Artificial Sequence Coding sequence for
peptide-based body surface coloring reagent 149 ggatccgacc
ctggctggca tctgtcttgg agccctgtac ctctgccgac tggcggcgca 60
ggcggtgcag gttggcacct gagctggtcc ccagtaccgc tgccgacggg cggcaccagc
120 acttctaaag caagcaccac cactaccagc tccaagacca ctaccacttc
ttccaaaacc 180 accacgacca cttctaagac ttctactact tctagcagct
ctaccggtgg taatacttct 240 cagctgagca ccggcagcgg cggtcagggt
ggcaatacgt ctcagctgtc caccggtggc 300 ccgaaaaagt aataaggcgc gcc 323
150 323 DNA Artificial Sequence Coding sequence for peptide-based
body surface coloring reagent 150 ggatccgacc ctggctggca tctgtcttgg
agccctgtac ctctgccgac tggcggcgca 60 ggcggtgcag gttggcacct
gagctggtcc ccagtaccgc tgccgacggg cggcaccagc 120 acttctaaag
caagcaccac cactaccagc tccaagacca ctaccacttc ttccaaaacc 180
accacgacca cttctaagac ttctactact tctagcagct ctaccggtgg taatacttct
240 cagctgagca ccggcagcgg cggtcagggt ggcaatacgt ctcagctgtc
caccggtggc 300 ccgaaaaagt aataaggcgc gcc 323 151 242 DNA Artificial
Sequence Coding sequence for peptide-based body surface coloring
reagent 151 ggatccgacc caggtactcc tccagaactg ctgcatggtg ctccacgttc
tggcggtgct 60 ggtggtgccg gcacccctcc agaactgctg cacggcgcac
cgcgctctgg cggtgcaggt 120 ggcgcagttt ggcacctgtc ctggtcccct
gtgccgctgc caaccggtgg cgcgggtggt 180 gctggttggc acctgagctg
gagcccggtt cctctgccga ccggtaaatg atgaggcgcg 240 cc 242 152 5388 DNA
Artificial Sequence Plasmid pKSIC4-HCC77623 152 agatctcgat
cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc 60
tagaaataat tttgtttaac tttaagaagg agatatacat atgcataccc cagaacacat
120 caccgccgtg gtacagcgct ttgtggctgc gctcaatgcc ggcgatctgg
acggcatcgt 180 cgcgctgttt gccgatgacg ccacggtgga agagcccgtg
ggttccgagc ccaggtccgg 240 tacggctgcg tgtcgtgagt tttacgccaa
ctcgctcaaa ctgcctttgg cggtggagct 300 gacgcaggag tgccgcgcgg
tcgccaacga agcggccttc gctttcaccg tcagcttcga 360 gtatcagggc
cgcaagaccg tagttgcgcc ctgtgatcac tttcgcttca atggcgccgg 420
caaggtggtg agcatccgcg ccttgtttgg cgagaagaat attcacgcat gccagggatc
480 cgatccgact ccgccgacga atgtactgat gctggcaacc aaaggcggtg
gtacgcattc 540 cacgcacaac catggcagcc cgcgccacac gaatgctgac
gcaggcaatc cgggcggcgg 600 caccccacca accaatgtcc tgatgctggc
tactaaaggc ggcggcacgc attctaccca 660 caaccatggt agcccgcgcc
atactaatgc agatgccggc aacccgggcg gtggtacccc 720 gccaaccaac
gttctgatgc tggcgacgaa aggtggcggt acccattcca cgcataatca 780
tggcagccct cgccacacca acgctgatgc tggtaatcct ggtggcggta agaagaaata
840 ataaggcgcg ccgacccagc tttcttgtac aaagtggttg attcgaggct
gctaacaaag 900 cccgaaagga agctgagttg gctgctgcca ccgctgagca
ataactagca taaccccttg 960 gggcctctaa acgggtcttg aggggttttt
tgctgaaagg aggaactata tccggatatc 1020 cacaggacgg gtgtggtcgc
catgatcgcg tagtcgatag tggctccaag tagcgaagcg 1080 agcaggactg
ggcggcggcc aaagcggtcg gacagtgctc cgagaacggg tgcgcataga 1140
aattgcatca acgcatatag cgctagcagc acgccatagt gactggcgat gctgtcggaa
1200 tggacgatat cccgcaagag gcccggcagt accggcataa ccaagcctat
gcctacagca 1260 tccagggtga cggtgccgag gatgacgatg agcgcattgt
tagatttcat acacggtgcc 1320 tgactgcgtt agcaatttaa ctgtgataaa
ctaccgcatt aaagcttatc gatgataagc 1380 tgtcaaacat gagaattctt
gaagacgaaa gggcctcgtg atacgcctat ttttataggt 1440 taatgtcatg
ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 1500
cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
1560 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
ttcaacattt 1620 ccgtgtcgcc cttattccct tttttgcggc attttgcctt
cctgtttttg ctcacccaga 1680 aacgctggtg aaagtaaaag atgctgaaga
tcagttgggt gcacgagtgg gttacatcga 1740 actggatctc aacagcggta
agatccttga gagttttcgc cccgaagaac gttttccaat 1800 gatgagcact
tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 1860
agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
1920 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
ctgccataac 1980 catgagtgat aacactgcgg ccaacttact tctgacaacg
atcggaggac cgaaggagct 2040 aaccgctttt ttgcacaaca tgggggatca
tgtaactcgc cttgatcgtt gggaaccgga 2100 gctgaatgaa gccataccaa
acgacgagcg tgacaccacg atgcctgcag caatggcaac 2160 aacgttgcgc
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 2220
agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
2280 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
tcattgcagc 2340 actggggcca gatggtaagc cctcccgtat cgtagttatc
tacacgacgg ggagtcaggc 2400 aactatggat gaacgaaata gacagatcgc
tgagataggt gcctcactga ttaagcattg 2460 gtaactgtca gaccaagttt
actcatatat actttagatt gatttaaaac ttcattttta 2520 atttaaaagg
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 2580
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
2640 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
taccagcggt 2700 ggtttgtttg ccggatcaag agctaccaac tctttttccg
aaggtaactg gcttcagcag 2760 agcgcagata ccaaatactg tccttctagt
gtagccgtag ttaggccacc acttcaagaa 2820 ctctgtagca ccgcctacat
acctcgctct gctaatcctg ttaccagtgg ctgctgccag 2880 tggcgataag
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 2940
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
3000 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
aagggagaaa 3060 ggcggacagg tatccggtaa gcggcagggt cggaacagga
gagcgcacga gggagcttcc 3120 agggggaaac gcctggtatc tttatagtcc
tgtcgggttt cgccacctct gacttgagcg 3180 tcgatttttg tgatgctcgt
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 3240 ctttttacgg
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 3300
ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
3360 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
tgatgcggta 3420 ttttctcctt acgcatctgt gcggtatttc acaccgcata
tatggtgcac tctcagtaca 3480 atctgctctg atgccgcata gttaagccag
tatacactcc gctatcgcta cgtgactggg 3540 tcatggctgc gccccgacac
ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 3600 tcccggcatc
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 3660
tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
3720 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
ttctccagaa 3780 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
ggcggttttt tcctgtttgg 3840 tcactgatgc ctccgtgtaa gggggatttc
tgttcatggg ggtaatgata ccgatgaaac 3900 gagagaggat gctcacgata
cgggttactg atgatgaaca tgcccggtta ctggaacgtt 3960 gtgagggtaa
acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc 4020
aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
4080 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
agactttacg 4140 aaacacggaa accgaagacc attcatgttg ttgctcaggt
cgcagacgtt ttgcagcagc 4200 agtcgcttca cgttcgctcg cgtatcggtg
attcattctg ctaaccagta aggcaacccc 4260 gccagcctag ccgggtcctc
aacgacagga gcacgatcat gcgcacccgt ggccaggacc 4320 caacgctgcc
cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg atggatatgt 4380
tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg gctccaattc
4440 ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
aggtggcccg 4500 gctccatgca ccgcgacgca acgcggggag gcagacaagg
tatagggcgg cgcctacaat 4560 ccatgccaac ccgttccatg tgctcgccga
ggcggcataa atcgccgtga cgatcagcgg 4620 tccagtgatc gaagttaggc
tggtaagagc cgcgagcgat ccttgaagct gtccctgatg 4680 gtcgtcatct
acctgcctgg acagcatggc ctgcaacgcg ggcatcccga tgccgccgga 4740
agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg ccagcaagac
4800 gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
cgaaacgttt 4860 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
aagattccga ataccgcaag 4920 cgacaggccg atcatcgtcg cgctccagcg
aaagcggtcc tcgccgaaaa tgacccagag 4980 cgctgccggc acctgtccta
cgagttgcat gataaagaag acagtcataa gtgcggcgac 5040 gatagtcatg
ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat 5100
cggtcgatcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
5160 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
gcgcccaaca 5220 gtcccccggc cacggggcct gccaccatac ccacgccgaa
acaagcgctc atgagcccga 5280 agtggcgagc ccgatcttcc ccatcggtga
tgtcggcgat ataggcgcca gcaaccgcac 5340 ctgtggcgcc ggtgatgccg
gccacgatgc gtccggcgta gaggatcg 5388 153 12 PRT Artificial Sequence
Hair conditioner and shampoo resistant hair-binding peptide 153 Gly
Met Pro Ala Met His Trp Ile His Pro Phe Ala 1 5 10 154 15 PRT
Artificial Sequence Hair conditioner and shampoo resistant
hair-binding peptide 154 His Asp His Lys Asn Gln Lys Glu Thr His
Gln Arg His Ala Ala 1 5 10 15 155 20 PRT Artificial Sequence Hair
conditioner and shampoo resistant hair-binding peptide 155 His Asn
His Met Gln Glu Arg Tyr Thr Asp Pro Gln His Ser Pro Ser 1 5 10 15
Val Asn Gly Leu 20 156 20 PRT Artificial Sequence Hair conditioner
and shampoo resistant hair-binding peptide 156 Thr Ala Glu Ile Gln
Ser Ser Lys Asn Pro Asn Pro His Pro Gln Arg 1 5 10 15 Ser Trp Thr
Asn 20
* * * * *