U.S. patent application number 11/876258 was filed with the patent office on 2008-07-31 for peptide derivatives, and their use for the synthesis of silicon-based composite materials.
This patent application is currently assigned to GENENCOR INTERNATIONAL, INC.. Invention is credited to Risha Lindig Bond, William Albert Cuevas, Joseph C. McAuliffe.
Application Number | 20080182971 11/876258 |
Document ID | / |
Family ID | 29584339 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080182971 |
Kind Code |
A1 |
McAuliffe; Joseph C. ; et
al. |
July 31, 2008 |
PEPTIDE DERIVATIVES, AND THEIR USE FOR THE SYNTHESIS OF
SILICON-BASED COMPOSITE MATERIALS
Abstract
Methods for forming peptide derivatives using functional
moieties and peptide derivatives are provided. Further, methods for
using peptide derivatives to form silicon-based composite materials
and silicon-based composite materials formed thereby are provided.
The silicon-based composite materials may have features on the
nanoscale, and the materials may exhibit characteristics derived
from the functional moieties on the peptide derivatives. It is
emphasized that this abstract is provided to comply with the rules
requiring an abstract which will allow a searcher or other reader
to quickly ascertain the subject matter of the technical
disclosure. It is submitted with the understanding that is will not
be used to interpret or limit the scope or meaning of the claims.
37 CFR 1.72(b).
Inventors: |
McAuliffe; Joseph C.;
(Sunnyvale, CA) ; Bond; Risha Lindig; (Menlo Park,
CA) ; Cuevas; William Albert; (San Francisco,
CA) |
Correspondence
Address: |
DINSMORE & SHOHL LLP
ONE DAYTON CENTRE, ONE SOUTH MAIN STREET, SUITE 1300
DAYTON
OH
45402-2023
US
|
Assignee: |
GENENCOR INTERNATIONAL,
INC.
Palo Alto
CA
|
Family ID: |
29584339 |
Appl. No.: |
11/876258 |
Filed: |
October 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10441908 |
May 20, 2003 |
7361731 |
|
|
11876258 |
|
|
|
|
60381928 |
May 20, 2002 |
|
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Current U.S.
Class: |
530/345 |
Current CPC
Class: |
B82Y 30/00 20130101;
B82Y 10/00 20130101; C07K 1/13 20130101; C07K 7/06 20130101; C07K
7/08 20130101; C07K 14/001 20130101; C07K 1/1077 20130101; C07K
14/405 20130101; C08G 77/452 20130101 |
Class at
Publication: |
530/345 |
International
Class: |
C07K 1/00 20060101
C07K001/00 |
Claims
1. A method of forming a composite material comprising: providing a
substantially pure peptide having at least two amino acids and less
than about 45 amino acids, wherein: at least one amino acid has a
polar functionality; modifying said peptide with a first functional
moiety selected from the group consisting of 1-pyreneacetic acid,
1-pyrenemethylamine, 5(6)-carboxyfluorescein, EDTA, cyclam
tetraacetic acid, lauric acid, cholesterol, D-biotin,
carboxymethyl-.beta.-cyclodextrin, and cysteine to form a peptide
derivative; and exposing said peptide derivative to a precursor
containing a silicon species such that a composite material forms,
wherein said peptide derivative and said silicon species are
incorporated into said composite material.
2. (canceled)
3. The method of claim 1 wherein said peptide has between about 7
to about 30 amino acids.
4. The method of claim 1 wherein said peptide is polybasic.
5. The method of claim 1 wherein said peptide has a pI of greater
than about 6.5.
6. The method of claim 1 wherein said peptide has a pI of between
about 7 to about 12.
7. The method of claim 1 wherein said peptide has a pI of between
about 8 to about 12.
8.-11. (canceled)
12. The method of claim 1 wherein said peptide derivative has
characteristics derived from said first functional moiety
13. The method of claim 1 wherein said polar functional amino acid
is selected from lysine, histidine, arginine, serine, tyrosine,
threonine, asparagine, glutamine and cysteine, and combinations
thereof.
14. The method of claim 1 wherein said polar functional amino acid
is selected from lysine, histidine, and arginine, and combinations
thereof.
15. The method of claim 1 wherein said peptide has at least one
motif comprising SGS wherein said motif is flanked by an amino acid
selected from a basic amino acid and an aromatic amino acid.
16. The method of claim 1 wherein said peptide has at least one
incidence of two or more tandem repeat polar functional amino
acids.
17. The method of claim 1 wherein said peptide is modified with a
plurality of functional moieties to form said peptide
derivative.
18. The method of claim 17 wherein said peptide is modified with
between 1 to 3 functional moieties.
19. The method of claim 1 wherein said first functional moiety is
selected from dyes, tracers, chemical indicators, fluorophores,
luminophores, biomolecules, biologically active compounds, enzymes,
liquid crystals, enzyme inhibitors, metal chelators, metal
complexes, nanoparticles, quantum dots, radioisotopes, cysteine or
drugs.
20. The method of claim 1 wherein said first functional moiety is
selected from 1-pyreneacetic acid and 1-pyrenemethylamine.
21. The method of claim 1 wherein said first functional moiety
comprises 5(6)-carboxyfluorescein.
22. The method of claim 1 wherein said first functional moiety
comprises EDTA.
23. The method of claim 1 wherein said first functional moiety
comprises cyclam tetraacetic acid.
24. The method of claim 1 wherein said first functional moiety
comprises lauric acid.
25. The method of claim 1 wherein said first functional moiety
comprises cholesterol.
26. The method of claim 1 wherein said first functional moiety
comprises D-biotin.
27. The method of claim 1 wherein said first functional moiety
comprises carboxymethyl-y-cyclodextrin.
28. The method of claim 1 wherein said first functional moiety
comprises cysteine.
29.-36. (canceled)
37. The method of claim 1 wherein said silicon containing species
is selected from Q-unit silanes, T-unit silanes, D-unit silanes,
and M-unit silanes.
38. The method of claim 1 wherein said silicon containing species
is selected from orthosilicic acid, tetramethoxysilane, and
tetraethoxysilane.
39. The method of claim 1 wherein said silicon containing species
is selected from phenyltriethoxysilane, phenyltrichlorosilane,
3-aminopropyltriethoxysilane, and methyltriemethoxysilane.
40. The method of claim 1 wherein said silicon containing species
is selected from phenylmethyldichlorosilane and
dimethyldimethoxysilane.
41. The method of claim 1 wherein said silicon containing species
comprises trimethylchlorosilane.
42. The method of claim 1 wherein said silicon containing species
is treated prior to exposing said peptide derivative to said
precursor containing said silicon species such that the silanol
content of said silicon species is maximized.
43. The method of claim 1 wherein said peptide derivative is
exposed to said precursor containing said silicon species occurs in
solution at a pH of about 5 to about 10.
44. The method of claim 43 wherein said solution has a pH of about
6 to about 9.
45. The method of claim 43 wherein said solution has a pH of about
7 to about 8.
46. The method of claim 1 further comprising forming an ordered
pattern on a substrate with said peptide derivative prior to
exposing said peptide derivative to said precursor containing said
silicon species.
47. The method of claim 46 wherein said ordered pattern is formed
by soft lithography.
48. The method of claim 46 wherein said ordered pattern is formed
by ink jet modified printing.
49. The method of claim 1 further comprising treating said
composite such that an organic portion of said composite is
altered.
50. The method of claim 1 wherein said composite material has
features on the nanoscale.
51. The method of claim 1 wherein said peptide derivative is
exposed to said precursor in the presence of an electric field.
52. The method of claim 1 wherein said peptide derivative is
exposed to said precursor in the presence of a magnetic field.
53. The method of claim 1 wherein said peptide derivative is
provided in a porous matrix, and wherein said peptide derivative is
exposed to said precursor in said porous matrix.
54. The method of claim 53 wherein said peptide derivative is
exposed to said precursor in the presence of an electric field.
55. The method of claim 53 wherein said peptide derivative is
exposed to said precursor in the presence of a magnetic field.
56.-96. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
application Ser. No. 10/441,908 filed May 20, 2003, which claims
priority to U.S. Provisional Application No. 60/381,928, filed May
20, 2002.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the formation of peptide
derivatives and to their use in the formation of functional
silicon-based composite materials.
[0003] Silicon-based materials, such as silica (SiO.sub.2) and
silicone resins, are used in a wide array of applications, and
there is growing interest in materials ordered at the nanoscale.
The ability to order silicon-based materials on a nanoscale with
organic templates such as polymers and surfactants provides
opportunities to produce organic-inorganic hybrid composite
materials having a variety of uses (Hou et al., Nature (1994), 368,
317-321).
[0004] Chemical synthesis of these materials generally requires
harsh conditions involving extremes of temperature or pH. It has
been recognized that amines and polyamines may catalyze the
polycondensation of silicic acid in water to form a silica
composite (Mizuntani et al., Bull. Chem. Soc. Jpn. (1998) 71,
2017-2022; Mizuntani et al., Chem. Lett. (1998), 133-134). More
recently, the problems of chemical synthesis have been addressed
using biological or biochemical synthesis techniques. The art has
recognized that certain proteins and peptides are able to produce
highly ordered biosilicates under ambient conditions (Zhou et al.,
Angew. Chem. Int. Ed. (1999) 38, 780-782). One particular class of
peptides, the silaffins which are found in diatoms (Kroger et al.,
Science (1999) 286, 1129-1132; Kroger et al., J. Biol. Chem. (2001)
276, 26066-26070) have been observed to produce silica nanospheres
and have recently been exploited in the production of optical
materials (Brott et al., Nature (2001) 413, 291-293).
[0005] There remains a need in the art to provide additional
silicon-based hybrid materials.
SUMMARY
[0006] The present invention meets that need by providing peptides
that have been modified with at least one functional group. The
peptides may be utilized as templates in the formation of
silicon-based hybrid materials. The resulting silicon-based hybrid
materials will have the functionality imparted by the functional
group or groups on the peptides.
[0007] In accordance with an embodiment of the present invention a
method of forming a composite material is provided. The method
comprises providing a peptide having at least two amino acids. At
least one amino acid has a polar functionality, and the peptide is
substantially pure. The method further comprises modifying the
peptide with a first functional moiety to form a peptide derivative
and exposing the peptide derivative to a precursor containing a
silicon species such that a composite material forms, wherein the
peptide derivative and the silicon species are incorporated into
said composite material.
[0008] In accordance with another embodiment of the present
invention a method of forming a peptide derivative is provided. The
method comprises providing a peptide having at least five amino
acids. At least one amino acid has a polar functionality. The
peptide has at least one motif comprising SGS, and the motif is
flanked by an amino acid selected from a basic amino acid or an
aromatic amino acid. The peptide is substantially pure. The method
further comprises modifying the peptide with a first functional
moiety to form a peptide derivative, wherein the peptide derivative
has characteristics derived from the first functional moiety.
[0009] In accordance with yet another embodiment of the present
invention a material comprising a composite material having a
peptide derivative portion and a silicon containing portion is
provided. The peptide derivative comprises a peptide modified with
a functional moiety, and the peptide comprises at least two amino
acids. At least one of said amino acids has a polar functionality.
The composite material exhibits a functionality derived from the
functional moiety.
[0010] In accordance with an embodiment of the present invention a
peptide derivative comprising a peptide modified with a functional
moiety is provided. The peptide has at least five amino acids, and
the peptide comprises at least one motif. The motif comprises SGS
flanked by an amino acid selected from a basic amino acid and an
aromatic amino acid containing species. The peptide has less than
about 45 amino acids, and the peptide has a pl greater than about
6.5.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] The following detailed description of the preferred
embodiments of the present invention can be best understood when
read in conjunction with the following drawings, in which:
[0012] FIGS. 1A-1H represent functional moieties that may be used
in embodiments of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013] The present invention involves the modification of peptides
to form peptide derivatives and the use of peptide derivatives to
produce composite materials having desired characteristics.
[0014] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth as used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless otherwise indicated, the
numerical properties set forth in the following specification and
claims are approximations that may vary depending on the desired
properties sought to be obtained in embodiments of the present
invention. Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from error
found in their respective measurements.
[0015] The peptides of the present invention are amino acid based
materials that contain a plurality of amino acids, for example, at
least 2, at least 5, or at least 7 amino acids. For example, the
peptides may have less than about 45 amino acids or about 7 to
about 30 amino acids. The amino acids may be the same repeating
amino acids, for example, polyarginine. The peptides may be
polypeptides including homopolymers. The peptides of the present
invention may generally contain amino acids having polar
functionality including lysine, histidine, arginine, serine,
tyrosine, threonine, asparagine, glutamine, glycine and cysteine.
These amino acids may bind to silicon through hydrogen bonding and
ionic interactions, and the polar amino acids thus facilitate the
formation of composites as discussed herein. For example, the
peptide chain may contain at least one basic amino acid selected
from lysine, histidine, and arginine or combinations thereof.
[0016] The peptides are generally peptides of defined amino acid
sequence, and, therefore, the peptides are substantially pure. For
purposes of defining and describing the present invention,
"substantially pure" shall be understood as referring to peptides
that comprise at least about 90% of a single peptide of defined
amino acid sequence. For example, the peptide may be about 95% or
about 97% of a single peptide of defined amino acid sequence. The
peptides may be individual substantially pure peptides or mixtures
thereof. In accordance with another embodiment of the present
invention, the peptides may be substantially monodispersed. The
term substantially monodispersed peptide means a peptide having a
narrow molecular weight distribution. By narrow molecular weight
distribution it is meant the peptides have a polydispersity of
Mw/Mn between 1.00 to 1.04. In another embodiment of the present
invention, the polydispersity is between 1.00 to 1.03. M.sub.n is
the number average molecular weight, and it is equal to
[.SIGMA.(N.sub.i)(M.sub.i)]/[.SIGMA.(N.sub.i)], where N.sub.i is
the number of molecules of molecular weight M.sub.i. M.sub.w is the
weight average molecular weight, and it is equal to
[.SIGMA.(N.sub.i)(M.sub.i).sup.2]/[.SIGMA.(N.sub.i)(M.sub.i)].
Molecular weight and polydispersity can be determined by tandem
GPC/light scattering in 0.1 M lithium bromide in dimethylformamide
at 60.degree. C. using dn/dc values (c=concentration) measured in
this solvent at .lamda..sub.0=633 nm.
[0017] In accordance with an embodiment of the present invention,
the peptides may contain at least one motif of
serine-glycine-serine (SGS) flanked by an amino acid selected from
a basic amino acid, such as lysine, arginine, and histidine, or an
aromatic amino acid. Flanked shall be understood as referring to
having an amino acid that may be a basic amino acid or an aromatic
amino acid adjacent to each S in the SGS motif. In accordance with
another embodiment of the present invention, the peptides may have
at least one incidence of two or more tandem repeat polar
functional amino acids. Tandem repeat amino acids shall be
understood as referring to the same amino acid occurring in
adjacent positions. It will be understood that the peptides may
also have the motif SGS and at least one incidence of two or more
tandem repeat amino acids having polar functionality. In accordance
with another embodiment of the present invention, the peptides are
polybasic. By polybasic it is meant the peptide comprises at least
two basic amino acid residues. For example, the peptides may have a
pl of greater than about 6.5. In a further example, the peptides
may have a pl of between about 7 to about 12. In another example,
the peptides may have a pl of between about 8 to about 12.
[0018] Examples of suitable peptides include, but are not limited
to, R5 (SEQ ID NO: 1), R2 (SEQ ID NO: 2), P1 (SEQ ID NO: 3), P2
(SEQ ID NO: 4), P3 (SEQ ID NO: 5), P4 (SEQ ID NO: 6), P5 (SEQ ID
NO: 7), R1 (SEQ ID NO: 16), R4 (SEQ ID NO: 17), Si3-3 (SEQ ID NO:
18), Si3-4 (SEQ ID NO: 19), Si3-8 (SEQ ID NO: 20), Si4-1 (SEQ ID
NO: 21), Si4-3 (SEQ ID NO: 22), Si4-7 (SEQ ID NO: 23), Si4-8 (SEQ
ID NO: 24), and Si4-10 (SEQ ID NO: 25).
[0019] R5 (SEQ ID NO: 1) has a sequence of SSKKSGSYSGSKGSKRRIL
(S=serine; K=lysine; G=glycine; Y=tyrosine; R=arginine;
I=isoleucine; L=leucine) and represents the backbone sequence of
the naturally occurring silaffin-1A.sub.1 peptide (Kroger et al.,
Science (1999) 286, 1129-1132). However, synthetic R5 (SEQ ID NO:
2) does not have lysine modifications as found in the naturally
occurring silaffin-1A.sub.1 from diatoms. R2 (SEQ ID NO: 2)
represents a variation on the backbone sequence of
silaffin-1A.sub.2, a naturally occurring peptide, has a sequence of
SSKKSGSYSGYSTKKSGSRIL (T=threonine) and differs from the naturally
isolated peptide in its lack of one arginine residue and the
posttranslational modifications of lysine. P1 (SEQ ID NO: 3) has a
sequence of LDAQERRRERRAEKQEQWKAAN (D=Aspartic Acid; A=alanine,
Q=Glutamine; E=Glutamic Acid; W=tryptophan; N=Asparagine) and is
derived from the RNA binding N-protein (Legault et al. Cell (1998)
93, 289-299). P2 (SEQ ID NO: 4) has a sequence of
SSHKSGSYSGSHGSHRRIL and is not a naturally occurring peptide. P3
(SEQ ID NO: 5) has a sequence of CSKKSGSYSGSKGSKRRCL, and P3 may be
cyclized or uncyclized. P4 (SEQ ID NO: 6) has a sequence of
SKKSGSKKSGSKKSGIL and is not a naturally occurring peptide. P5 has
a sequence of RRRRRRRRR (SEQ ID NO: 7) and is modified by Ahx to be
Ahx-RRRRRRRRR (Ahx=2-aminohexanoic acid).
[0020] R1 has a sequence of SSKKSGSYYSYGTKKSGSYSGYSTKKSASRRIL (SEQ
ID NO: 16) and represents the backbone sequence of the naturally
occurring silaffin peptide (Kroger et al., Science (1999) 286,
1129-1132). R4 has a sequence of SSKKSGSYSGSKGSKRRNL (SEQ ID NO:
17) and represents the backbone sequence of the naturally occurring
silaffin peptide (Kroger et al., Science (1999) 286,
1129-1132).
[0021] Si3-3 has a sequence of APPGHHHWHIHH (SEQ ID NO: 18). Si3-4
has a sequence of MSASSYASFSWS (SEQ ID NO: 19). Si3-8 has a
sequence of KPSHHHHHTGAN (SEQ ID NO: 20). Si4-1 has a sequence of
MSPHPHPRHHHT (SEQ ID NO: 21). Si4-3 has a sequence of MSPHHMHHSHGH
(SEQ ID NO: 22). Si4-7 has a sequence of LPHHHHLHTKLP (SEQ ID NO:
23). Si4-8 has a sequence of APHHHHPHHLSR (SEQ ID NO: 24). Si4-10
has a sequence of RGRRRRLSCRLL (SEQ ID NO: 25). Si3-3 to Si4-10
(SEQ ID NO: 18-25) are random 12 amino acid peptides derived from a
combinatorial library (Naik et al., J. Nanosci. Nanotech., 2002,
Vol. 2, No. 1, 95-97).
[0022] In accordance with another embodiment of the present
invention, a portion of the primary structure of the sill p protein
may be used as the peptides of the present invention. Silp1 has a
sequence of: MKLTAIFPLLFTAVGYCMQSIADLAAANLSTEDSKSAQLISADSSDDASDSSVE
SVDMSSDVSGSSVESVDVSGSSLESVDVSGSSLESVDDSSEDSEEEELRILSS
KKSGSYYSYGTKKSGSYSGYSTKKSASRRILSSKKSGSYSGYSTKKSGSRRILS
SKKSGSYSGSKGSKRRILSSKKSGSYSGSKGSKRRNLSSKKSGSYSGSKGSK
RRILSSKKSGSYSGSKGSKRRNLSSKKSGSYSGSKGSKRRILSGGLRGSM (SEQ ID NO: 26)
(Kroger et al., Science (1999) 286, 1129-1132). Subfragments of the
sill P sequence having at least 2, at least 5, or at least 7 amino
acids may be used in accordance with the present invention.
[0023] The peptides of the present invention are generally produced
according to well known synthetic methods (Fields, G. B. (ed.)
Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis
(1997) Academic Press). For example, the peptides may be produced
using standard solid-phase chemistry on an automated peptide
synthesizer, such as an Applied Biosystems (Foster City, Calif.)
433A automated peptide synthesizer.
[0024] The peptides of the present invention are modified with at
least one functional moiety, for example, a single or a plurality
of functional moieties, to form a peptide derivative. As used
herein, the term "modified" is defined to mean the covalent
attachment of at least one functional moiety to a peptide at a
predefined location. As used herein, the term "predefined location"
is defined to mean a specific desired residue position within the
peptide. For example, pyrene moieties may be attached to the two
glutamines of SEQ ID NO: 3. As used herein, the term "functional
moiety" is defined to include any species that imparts its
characteristics to the molecule to which it is attached, including
the impartation of chemical or physical behaviors. Therefore, the
peptide derivative may have characteristics derived from the
functional moiety, as may any resulting material incorporating the
peptide derivative. Desirable functional moieties include, but are
not limited to, dyes, tracers, chemical indicators, fluorophores,
luminophores, biomolecules, biologically active compounds, enzymes,
liquid crystals, enzyme inhibitors, metal chelators, metal
complexes, nanoparticles, quantum dots, radioisotopes, drugs and
the like. Additionally, amino acids that may influence the
structure of the peptide and act in a functional manner may be
functional moieties. For example, cysteine has the ability to allow
the peptide to be cyclized and may act as a metal chelator. It will
be understood that any functional moiety may be used for which a
suitable chemical method for covalently attaching the functional
moiety to the peptide exists. Alternatively, any functional moiety
may be used for which a suitable biological method for covalently
attaching the functional moiety to the peptide exists. Some
examples of suitable biological and chemical methods are provided
herein. In accordance with one embodiment, the functional moieties
are attached to the peptides by solid phase chemistry.
[0025] The peptides of the present invention may generally be
derivatized with at least one functional moiety. For example, the
peptides may contain one to three functional moieties. When the
peptide contains at least two functional moieties, the first
functional moiety may be the same as or different from the
subsequent functional moieties. Additionally, the first functional
moiety may have the same or a different function than the
subsequent functional moieties. The functional moieties may be
attached to any amino acid in the peptide through methods detailed
in the art (Hermanson G. T., Bioconjugate Techniques (1996)
Academic Press).
[0026] For example, suitable functional moieties include
fluorophores such as pyrene and fluorescein. Other suitable
fluorophores may be found in the Handbook of Fluorescent Probes and
Research Products, 9.sup.th Ed (Molecular Probes, Eugene, Oreg.).
Labeling the peptide with a fluorophore such as pyrene or
fluorescein alters the optical properties of the peptide and also
may influence the morphology of composites derived from the peptide
derivative. The optical properties of the fluorophore and the
influence of this moiety on the morphology of the nanocomposites
are not necessarily related. The peptide may be labeled using
1-pyreneacetic acid as shown in FIG. 1A or 5(6)-carboxyfluorescein
as shown in FIG. 1B. 1-pyrenemethylamine may also be used to label
the peptide.
[0027] For example, R5 (SEQ ID NO 1) may have pyrene or fluorescein
labels attached to the N-terminus. Similarly, P1 (SEQ ID NO 3) may
have pyrene labels on the glutamates. The labeled glutamates may be
LDAQERRRERRAEKQEQWKAAN (SEQ ID NO: 3) where the labeled glutamates
are indicated by underlining. Similarly, a fluorescein label may be
attached to the N-terminus of an Ahx modified P5 (SEQ ID NO 7), and
composites derived from this peptide derivative may be useful in
gene and protein delivery to cells because the peptide derivative
has the ability to traverse cell membranes (Futaki et al.
Bioconjugate. Chem. (2001) 12, 1005-1011).
[0028] Other suitable functional moieties include enzymes such as
subutilisin or .beta.-lactamase. Once the peptide-enzyme derivative
has been incorporated into a composite material, the composite may
posses enzymatic activity. For example, subtilisin may be attached
to the R5 peptide (SEQ ID NO 1). Similarly, the R5 peptide (SEQ ID
NO 1) could be attached to the enzyme .beta.-lactamase.
[0029] Another suitable functional moiety includes moieties that
may impart hydrophobic or amphiphilic functionality. For example,
saturated or unsaturated long chain fatty acids (C.sub.6-C.sub.22)
may be used. One such fatty acid is lauric acid as shown in FIG.
1E. Perhydrocyclopentaphenanthrene derivatives may also provide the
function of increased hydrophobicity. Steroids with 8-10 carbon
atoms in the side chain at position 17 and an alcoholic hydroxyl
group at position 3 are also suitable. For example cholesterol, as
shown in FIG. 1F, is a suitable steroid (White, et al Principles of
Biochemistry, Fifth Edition, pp 78-85). For example, the moieties
may modify the physical properties, such as surfactant properties
or the physical morphology, of the peptide derivative and resulting
composite materials. Lauric acid may be attached to the P4 peptide
(SEQ ID NO 6). In a further example, the N-terminus of P4 (SEQ ID
NO 6) may be labeled with cholesterol.
[0030] Suitable functional moieties include chelating agents. For
example, suitable chelating agents include, but are not limited to
porphyrins such as, porphine, heme and chlorophyll; vitamin B12,
and dimercapol. Other suitable chelating agents include cyclam
tetraacetic acid, as shown in FIG. 1H, and EDTA as shown in FIG.
1C. The chelating agents may impart metal chelating activity to the
peptide derivatives. For example, cyclam tetraacetic acid may be
added to the R5 (SEQ ID NO 2) peptide to produce a peptide
derivative with metal chelating activity.
[0031] Another functional moiety may impart a protein binding
ability as a possible site for the attachment of proteins.
D-biotin, as shown in FIG. 1D, may be a suitable functional moiety,
and the D-biotin is a known ligand for proteins (biotin binding
proteins, for example, avidin and/or streptavidin). For example,
the N-terminus of P4 (SEQ ID NO 6) may be labeled with D-biotin.
Carboxymethyl-.beta.-cyclodextrin, as shown in FIG. 1G, may be a
suitable functional moiety, and carboxymethyl-.beta.-cyclodextrin
may provide the peptide derivative with the ability to encapsulate
hydrophobic guest molecules (D'Souza, V. T., Lipkowitz, K. B.
Chemical Reviews (1998), 98, 1741-1742). For example,
carboxymethyl-.beta.-cyclodextrin may be added to the R5 peptide
(SEQ ID NO 1).
[0032] The functional moieties may be added to the peptides using
chemical or biological methods. For example, the functional
moieties may be added chemically while the peptide is still on the
resin after automated peptide synthesis. The substitution of the
peptide on the resin is generally calculated manually or by using
software, such as software available under the tradename
SYNTHASSIST.RTM. software from Applied Biosystems (Foster City,
Calif.). The groups protecting the amino acids to be substituted
are removed, and the resin is swelled in a solvent such as
N-methyl-2-pyrrolidone (NMP) prior to the addition of the precursor
containing the functional moiety. The functional moiety is added to
the resin slurry, and the reaction is allowed to proceed. The
reaction may be promoted by additional reagents or catalysts,
including enzymes, depending on the nature of the desired chemical
functionality linking the peptide and the functional moiety. The
nature of these chemical functionalities includes but is not
limited to amides, esters, acetals, ketals, ethers, amines,
thioethers, thioesters, imines, phosphate esters, carbon-carbon
bonds, silicon-carbon bonds, silicon-oxygen bonds and the like.
After the reaction, the solid phase is typically washed, and the
modified peptide is cleaved, deprotected, and purified in
accordance with well-known methods. However, the functional
moieties may be added after cleavage and deprotection of the
peptide. In this instance, the unprotected peptide is dissolved in
a suitable solvent and attached to the functional moiety in a
similar fashion as described for resin-bound peptides. If multiple
products result from such treatment then one can improve the
chemical selectivity of the coupling reaction through methods
described in the art (Hermanson G. T., Bioconjugate Techniques
(1996) Academic Press) or apply a suitable technique for
purification of the desired conjugate following the reaction.
[0033] Alternatively, the entire peptide derivative comprising a
peptide and at least one functional moiety may be generated using
molecular biology techniques. This approach is particularly useful
for attaching a functional moiety such as a protein. In this
approach, a DNA sequence encoding the peptide is inserted into the
DNA sequence of the desired functional moiety. The insertion of a
DNA sequence encoding the peptide into a DNA sequence encoding the
desired functional moiety may be accomplished using well known
vector and fusion techniques. The peptide may then be expressed by
inserting the recombinant DNA into a host cell for replication and
expression. U.S. Pat. No. 5,679,543, the disclosure of which is
herein incorporated by reference, contains a number of references
to articles that outline suitable recombinant DNA techniques.
Additionally, Jeremy Thorner et al., Applications of chimeric genes
and hybrid proteins: Part A: Gene Expression and Protein
purification (Methods in Enzymology, vol. 326) (2000) contains
suitable methods for forming fusion proteins and is incorporated by
reference herein.
[0034] Once the peptide derivative has been formed, it is exposed
to a precursor containing a silicon species, and the peptide
derivative acts as a template in the formation of a silicon-based
composite. Ordinarily, the peptide derivative does not serve as a
catalyst. Rather, the peptide derivative becomes incorporated into
the composite to form a hybrid material comprising the peptide
derivative and the silicon containing species. The composite
material may be nanostructured in the form of nanoparticles or
aggregates thereof. Nanoparticles are distinct clusters or spheres
of material of diameter between about 1 and about 1000 nm. Other
morphologies are also possible however, including fibers,
laminates, gels, crystalline materials, porous solids and materials
with features on several distinct length scales from nanometers to
centimeters.
[0035] The silicon species in the precursor may be in any suitable
form. For example, silicates or organosilanes may be the silicon
species. For example, the silicon species may be in the form of a
Q-, T-, D- or M-unit silicate and silane or mixtures thereof.
Q-unit silanes have a silicon-containing group of the general
structure SiO.sub.4-- (four points of attachment). T-unit silanes
have a silicon-containing group of the general structure
--RSiO.sub.3-- (three points of attachment) where R represents any
group containing carbon. D-unit silanes have a silicon-containing
group of the general structure --R.sub.2SiO.sub.2-- (two points of
attachment). M-unit silanes have a silicon-containing group of the
general structure --R.sub.3SiO--. Examples of suitable precursors
include, but are not limited to inorganic Q units such as
orthosilicic acid (Si(OH).sub.4), its salts and oligomers, organic
Q units such as tetramethoxysilane (TMOS), tetraethoxysilane
(TEOS), T-units such as phenyltriethoxysilane,
phenyltrichlorosilane, 3-aminopropyltriethoxysilane,
methyltrimethoxysilane, D-units such as phenylmethyldichlorosilane,
dimethyldimethoxysilane and M-units such as trimethylchlorosilane.
These silane precursors may be pretreated to maximize the silanol
(Si--OH) content through either chemical or enzymatic hydrolysis.
For example, treatment of tetraethoxysilane (TEOS) in 1 mM
hydrochloric acid (HCl) results in a solution of orthosilicic acid
over several hours suitable for composite preparation.
[0036] The peptide derivative is generally exposed to the silicon
precursor in solution at a pH of between about 5 to about 10. For
example, a pH of between about 6 to about 9 may be used. In a
further example, a pH between about 7 to about 8 may be used. The
peptide derivative is generally exposed to the silicon precursor at
ambient temperature and pressure.
[0037] The peptide derivative may be exposed to the silicon
precursor solution in bulk. The peptide derivative may
alternatively be exposed to the silicon precursor by slow addition
or addition under dilute conditions in order to alter the
morphology of nanoparticles. In another alternative, the peptide
derivative may be exposed to the silicon precursor in the presence
of a suitable surfactant in order to alter the morphology of
nanoparticles. Additionally, the Stober process may be utilized to
promote monodispersity of, prevent aggregation of, or otherwise
alter the morphology of nanoparticles (Stober et al., Stober
Process for Controlled Particle Growth, E. J. Colloid Interface
Sci., 26, 62 (1968)). Alternately, the peptide derivative and
silane precursor may be mixed in a two phase system comprising two
immiscible solvents.
[0038] For example, the exposure of peptide derivatives in solution
to a silicic acid solution may produce a composite material of
silica and peptide, which may be in the form of a gel or solid
material. By "gel or solid" it is meant a gel or solid being about
50% or less aqueous or organic solvent by weight. The composite
material may also be in the form of aggregates, fibers, laminates,
and the like. In a further example, the exposure of the peptide
derivatives to an organosilane such as a T-, D-, or M-unit silane
may produce a composite material of organosilane and peptide
derivative. The composites may be 3-dimensional networks containing
organosilane units and peptide units. Such composite materials may
be useful in the formation of thin-films, coatings, and the like.
Thus, the composite materials may be hybrid materials that have
both inorganic and organic components.
[0039] Further treatment of the composite may provide new materials
wherein the organic portion of the composite is altered. For
example, the organic portion may be crosslinked or removed.
Exemplary methods of alteration include electromagnetic
irradiation, thermal treatment and/or chemical treatment. For
example, a composite could be constructed containing a reactive
functionality. Such functionality might originate from either a
modified peptide template or the silane precursor. Subsequent
crosslinking of the reactive functionality could result from
treatment of the composite by irradiation, chemical or thermal
treatment. Another example might involve the removal of all or part
of the organic portion of a composite by high temperature thermal
treatment (i.e. calcination). Such treatment could result in the
formation of composites with increased porosity and/or altered
morphology as compared to the untreated composites.
[0040] It may be possible to form patterned structures by using the
peptide derivative to form a pattern on any suitable substrate and
exposing the pattern to the silicon-containing precursor. Soft
lithography is a non-photolithographic technique useful for
carrying out micro- and nanofabrication. Soft lithography may
produce patterns and structures having feature sizes ranging from
about 30 nm to about 100 .mu.m. Soft lithography generally utilizes
an elastomeric stamp or mold with patterned relief structures on
its surface used to generate the desired pattern. In one
embodiment, an elastomeric stamp may be formed using a master mold.
The stamp is "inked" with the peptide derivative in a solution and
a substrate is contacted with the stamp. A pattern of peptide
derivative is formed on the substrate in the areas where the relief
structures of the stamp contacted the substrate. Examples of
suitable soft lithographic stamps are found in published U.S.
Patent Application Nos. 20010027570 and 20010013294, the
disclosures of which are incorporated by reference herein.
Alternatively, a mold may be formed and placed in contact with a
substrate. A peptide derivative solution is then placed at one end
of the mold, and channels in the mold fill by capillary action to
form a pattern after the mold is removed. Additionally, the
substrate itself may be patterned by soft lithography, and the
peptide derivative may then be applied to the substrate to fill the
pattern. For example, placing a mold on the substrate and filling
it with a prepolymer may pattern the substrate. U.S. Pat. No.
6,368,877 discloses several methods of forming patterns using soft
lithography and is incorporated by reference herein.
[0041] In rapid printing, a self assembling "ink" comprising the
peptide derivative in solution is used with rapid printing
procedures to form patterned structures in a very short period of
time. Suitable rapid printing procedures include pen lithography,
ink-jet printing, and dip-coating. The rapid printing procedures
use the ink to form a desired pattern on suitable substrates. The
ink thus forms patterned peptide derivatives that define
functional, hierarchically organized structures in seconds.
Suitable rapid printing techniques and apparatus are described in
Hongyou Fan, Rapid Prototyping of Patterned Functional
Nanostructures, Nature 405, 56-60 (2000), which is incorporated by
reference herein. Three-dimensional structures may be formed on a
suitable substrate by forming the peptide pattern, exposing the
pattern to a silicon-containing precursor, and repeating the
procedure until the desired structure has been achieved.
[0042] In accordance with another embodiment of the present
invention, the nanocomposites of the present invention may be
formed in an electric or magnetic field to provide control over the
morphology of the nanocomposite materials. Additionally, the
nanocomposites may be formed in a porous matrix to provide control
over the morphology of the nanocomposite. The peptide derivative
may be exposed to a suitable precursor in the presence of any
suitable electric or magnetic field. For example, the peptide
derivative may be provided in an agarose matrix and standard gel
electrophoresis equipment may be used to provide an electric field
during the exposure of the peptide derivative. In a further
example, a peptide derivative with a metal-chelating group may be
attached to magnetic particles, and the nanocomposite formation may
be performed in an electric field. For example, the magnetic
particles may be pulled through a silicate solution at an
appropriate pH. The electromagnetic parameters and
peptide/functional moieties may all be controlled to direct the
morphology of the resulting nanocomposites.
[0043] In order that the invention may be more readily understood,
reference is made to the following examples, which are intended to
be illustrative of the invention, but are not intended to be
limiting in scope.
Example 1
[0044] The R5 (SEQ ID NO: 1), P4 (SEQ ID NO: 6), and P1 (SEQ ID NO:
3) base sequences were created using standard Fmoc chemistry on an
Applied Biosystems 433A automated peptide synthesizer. For each
base sequence, 137 mg of ABI preloaded Fmoc Wang (HMP) resin was
used. Subsequent offline cleavage and deprotection of 100 mg resin
with attached peptide was performed in a cleavage solution
contained 1110 .mu.L trifluoroacetic acid (TFA), 30 .mu.L water, 30
.mu.L triisopropylsilane (TIS), and 30 .mu.L 1,2-ethanedithiol
(EDT), for a total volume of 1200 .mu.L. The reaction was allowed
to run 3-4 hours, and the deprotected peptide was then filtered
from the resin into 10 mL ice-cold (0.degree. C.) methyl-tert-butyl
ether (MtBE). The peptide was centrifuged in MtBE at 4900 rpm for 5
minutes, the MtBE poured off, and the peptide then resuspended in
fresh MtBE. This was cycle was repeated four times and the peptide
was then allowed to dry and submitted for HPLC analysis.
Purification was performed by preparative HPLC using a Vydac C18
column (22 mm by 250 mm) and eluted with a gradient of water (0.1%
TFA) and acetonitrile (0.08% TFA). Fractions containing the desired
material were pooled and lyophilized to yield the pure peptide.
Identity was confirmed by mass spectrometry.
Example 2
[0045] The P3 (SEQ ID NO: 5) peptide sequence was cyclized. The P3
peptide was synthesized using normal Fmoc chemistry on an automated
peptide synthesizer in accordance with Example 1. P3 peptide (65.6
mg) was cleaved from the resin and then cyclized by forming the
cysteine-cysteine disulfide bridge. This cyclization was induced
using EKATHIOX resin, made by Ekagen (Menlo Park, Calif.) and
distributed by Sigma-Aldrich (St. Louis, Mo.). A ten-fold molar
excess of resin active group (0.35 mmol/g, 1.0 gram EKATHIOX) was
stirred with the peptide in 33 mL deionized water with 0.5% (v/v)
acetic acid for approximately 48 hours. The EKATHIOX was then
filtered from the solution and the peptide was lyophilized.
Cyclization was confirmed by MALDI-TOF mass spectrometric analysis,
including a 50/50 mixture of treated and untreated P3 showing two
corresponding peaks
Example 3
[0046] Labeling of the N-terminus of the R5 (SEQ ID NO: 1) sequence
with fluorescein was performed while the peptide was still on the
resin and its side chain amino acid groups were still protected,
herein referred to as R5-resin. The final Fmoc removal from the
N-terminus was performed on the automated peptide synthesizer. The
substitution of peptide on resin was calculated by Applied
Biosystems Synthassist.RTM.software software, and 105 mg R5-resin
contained 23.1 .mu.mol peptide. The 105 mg of R5-resin was swollen
in 500 .mu.L N-methylpyrrolidone (NMP) for five minutes in a
fritted filtration vessel. About 25 equivalents of
diisopropylethiamine (DIEA), 285 .mu.L (2 M) DIEA in NMP, was added
to the R5-resin, and then 70 mg (152 .mu.mol) 5-carboxyfluorescein
was added. The reaction was protected from light and allowed to mix
in excess of 24 hours. The solid phase was then washed twice with
NMP and four times with dichloromethane (DCM) before being dried
under nitrogen. The peptide was cleaved and deprotected as
described in Example 1.
Example 4
[0047] Labeling of the N-terminus of the R5 (SEQ ID NO: 1) sequence
with pyrene was performed while the peptide was still on the resin
and its side chain amino acid groups were still protected, herein
referred to as R5-resin. The final Fmoc removal from the N-terminus
was performed on the automated peptide synthesizer. The
substitution of peptide on resin was calculated by software sold
under the tradename SYNTHASSIST.RTM. software by Applied Biosystems
(Foster City, Calif.), and 100 mg R5-resin contained 22.6 .mu.mol
peptide. The 100 mg of R5-resin was swollen in 500 .mu.L NMP for
five minutes in a fritted filtration vessel. Concurrently, 35.3 mg
(135.6 .mu.mol) pyreneacetic acid (PAA) was dissolved in 1 mL
dimethylsulfoxide (DMSO). After swelling of the resin, 250 .mu.L
(0.5 M) HBTU/HOBt solution was added to the R5-resin and allowed to
mix for 10 minutes. The next step was to add 150 .mu.L (2 M) DIEA
to the pyreneacetic acid solution. The PAA solution containing DIEA
was added to the R5-resin slurry and the reaction mixed for 20-30
minutes. The solid phase was then washed twice with DMSO, twice
with NMP, and three times with DCM before being dried under
nitrogen. The peptide was cleaved and deprotected as described in
Example 1.
Example 5
[0048] Two glutamatic acid residues of P1 (SEQ ID NO: 3) were
labeled with pyrene. The automated synthesis of P1 (SEQ ID NO: 3)
used two glutamatic acid residues protected by
2-phenylisopropylester (PiPE) groups and retained the Fmoc on the
N-terminus. The PiPE groups were removed by mixing 300 mg P1-resin
with a solution of 2% TFA and 5% TIS in DCM. The P1-resin was mixed
three times with 3 mL of deprotecting solution for 3-4 minutes each
time. A fritted filtering vessel was used to expedite this process.
The solid phase was then washed twice with 2% TIS in DCM and three
times with a 50/50 solution of DCM and methanol. After drying under
nitrogen, the PiPE-deprotected P1-resin was transferred to a round
bottom flask. The calculated substitution of the P1-resin was 0.139
.mu.mol/mg, thus the 300 mg P1-resin contained approximately 83.4
.mu.mol peptide. After swelling the resin in dimethylformamide
(DMF), 17.4 mg (129 .mu.mol) HOBt and 66.4 mg (128 .mu.mol) pyBOP
(NovaBiochem) were dissolved into the slurry. Then 250 .mu.L (2 M)
DIEA in NMP was added to the P1-resin mixture, and concurrently
113.2 mg (423 .mu.mol) 1-pyrenemethylamine (PMA) was dissolved
separately in DMSO. Both flasks were allowed to stir 10 minutes,
and then the PMA in DMSO was added to the activated P1-resin. The
final mixture was allowed to react for over an hour and then
transferred to a fritted filtration vessel. The solid phase was
washed twice with DMF, twice with DMSO, and four times with DCM,
and then dried under nitrogen. The labeled glutamates are
underlined in the labeled P1 (SEQ ID NO: 3) sequence
LDAQERRRERRAEKQEQWKAAN.
[0049] The Fmoc on the N-terminus was removed by reacting the
pyrene-labeled P1-resin in a solution of 20% piperidine in DMF for
1 hour. The solid phase was washed three times with DMF, twice with
a 50/50 solution of DCM and methanol, and three times with DCM. The
peptide was cleaved and the remaining side-chain protecting groups
removed as described in Example 1.
Example 6
[0050] The N-terminus of the P4 (SEQ ID NO: 6) sequence was labeled
with lauric acid. The labeling was performed while the peptide was
still on the resin and its side chain amino acid groups were still
protected, herein referred to as P4-resin. The final Fmoc removal
from the N-terminus was performed on the automated peptide
synthesizer. Substitution of peptide on resin was calculated by
Applied Biosystems software. P4-resin (83 mg, 22.7 .mu.mol peptide)
was swollen in 500 .mu.L NMP for five minutes. Concurrently, 28 mg
(137 .mu.mol) lauric acid, CH.sub.3(CH.sub.2).sub.10COOH, was
dissolved in 1 mL dimethylformamide (DMF), and then 1300 .mu.L (0.1
M in DMF) HBTU/HOBt solution was added to the lauric acid. The next
step was to add 150 .mu.L (2 M) DIEA to the lauric acid mixture and
allow it to stir. Then the solution containing lauric acid,
HBTU/HOBt, and DIEA was added to the P4-resin slurry and the
reaction mixed for at least one hour. The solid phase was then
washed twice with NMP, twice with DMF, and four times with
dichloromethane (DCM) before being dried under nitrogen. The
peptide was cleaved and deprotected as described in Example 1.
Example 7
[0051] The N-terminus of the P4 (SEQ ID NO: 6) sequence is labeled
with carboxymethyl .beta.-cyclodextrin (CM.beta.CD). The labeling
is performed while the peptide is still on the resin and its side
chain amino acid groups are still protected, herein referred to as
P4-resin. The final Fmoc removal from the N-terminus is performed
on the automated peptide synthesizer. Substitution of peptide on
resin is given to be 0.22 .mu.mol/mg. A solution of CM.beta.CD (263
mg, 220 .mu.mol), HOBt (28.5 mg, 211 .mu.mol),
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate (pyBOP) (114.5, 220 .mu.mol), and 500 .mu.L
(2M) DIEA in 9 mL DMSO is allowed to stir for one week.
Alternatively, a solution containing cyclam tetraacetic acid or
5(6)-carboxyfluorescein may be used. Then P4-resin (200 mg, 44
.mu.mol peptide) is swelled in 1 mL NMP and is added to the
solution containing CM.beta.CD. This final slurry is stirred
overnight. The solid phase is then washed twice with DMSO, twice
with DMF, and four times with DCM before being dried under
nitrogen. The peptide is cleaved and deprotected as described in
Example 1.
Example 8
[0052] The R5 sequence (SEQ ID NO: 1) was labeled with pyrene. In
this instance, the automated synthesis of R5 (SEQ ID NO: 1) used
two lysines protected by methyltrityl (Mtt) groups and retained the
Fmoc on the N-terminus. The Mtt groups were removed by stirring
R5-resin in three batches of 2 mL TFA-TIS solution (1%
trifluoroacetic acid, 3% triisopropylsilane in DCM) for five
minutes each batch. A fritted filtering vessel was used to expedite
this process. The solid phase was then washed twice with 2% TIS in
DCM and three times with a 50/50 solution of DCM and methanol.
After drying under nitrogen, the Mtt-deprotected R5-resin was
transferred to a round bottom flask. The Applied Biosystems
software gave a calculated substitution of 0.20 .mu.mol/mg. Thus,
the 90 mg R5-resin contained approximately 18 .mu.mol peptide, or
36 .mu.mol deprotected lysine sites. The resin was swollen in 500
.mu.L N-methylpyrrolidine (NMP). In a separate flask, 57.1 mg (216
.mu.mol) pyreneacetic acid (PAA) was dissolved in DMSO. First 2016
.mu.L HBTU/HOBt (0.1 M) and then 500 .mu.L DIEA (2M in NMP) was
added to the dissolved PAA and allowed to react for 10 minutes. The
solution containing PAA, HBTU/HOBt, and DIEA was added to the
R5-resin slurry.
[0053] The final mixture was allowed to react for over an hour and
then transferred to a fritted filtration vessel. The solid phase
was washed three times with DMSO, twice with NMP, and four times
with DCM, and then dried under nitrogen. Labeled lysines are
underlined in the R5 sequence: SSKKSGSYSGSKGSKRRIL (SEQ ID NO: 1).
The Fmoc may be removed from the N-terminus of the labeled peptide
in a reaction solution of 20% piperidine in DMF. The peptide may be
cleaved in accordance with the procedure of Example 1.
Example 9
[0054] The N-terminus of the P4 peptide (SEQ ID NO: 6) was labeled
with cholesterol. The labeling of the N-terminus of the P4 peptide
(SEQ ID NO: 6) was performed while the peptide was still on the
resin and its side chain amino acid groups were still protected,
herein referred to as P4-resin. The final Fmoc removal from the
N-terminus was performed on the automated peptide synthesizer. The
substitution of peptide on resin was estimated to be 0.20
.mu.mol/mg, and 140 mg P4-resin contained approximately 28 .mu.mol
peptide. The 140 mg of P4-resin was mixed with 152 mg (280/mol)
cholesterol chloroformate and 420 .mu.L (2 M) diisopropylethlamine
(DIEA) in NMP, in 8-10 mL NMP total. The reaction was allowed stir
at room temperature protected for 24 hours. The solid phase was
then washed twice with NMP and four times with dichloromethane
(DCM) before being dried under nitrogen. The peptide was cleaved
and deprotected as described in Example 1. The P4-cholesterol was
purified and its identity confirmed by MALDI-TOF mass
spectrometry.
Example 10
[0055] The N-terminus of the P4 peptide (SEQ ID NO: 6) was labeled
with EDTA dianhydride in accordance with the following procedure.
Labeling of the N-terminus of the P4 sequence (SEQ ID NO: 6) was
performed while the peptide was still on the resin and its side
chain amino acid groups were still protected, herein referred to as
P4-resin. The P4-resin used in this experiment was synthesized by
SynPep Corp. (Dublin, Calif.), lot 02GE2271, and the final Fmoc had
already been removed from the N-terminus. Substitution of the
peptide on resin was given to be 0.22 .mu.mol/mg. The
ethylenediaminetetraacetic acid (EDTA) dianhydride was added at
approximately five equivalents to the peptide. P4-resin (100 mg, 22
.mu.mol peptide) was swollen in N-methylpyrrolidone (NMP) and to
this was added 25 mg EDTA (100 .mu.mol) and 100 .mu.L (2M in NMP)
DIEA. The reaction was stirred for two hours and then quenched with
water. The solid phase was washed twice with NMP and four times
with DCM before being dried under nitrogen. The peptide-EDTA
conjugate was deprotected and cleaved as described in Example 1,
and the identity of the material confirmed by mass
spectrometry.
Example 11
[0056] The N-terminus of the P4 peptide (SEQ ID NO: 6) was labeled
with biotin in accordance with the following procedure. Biotin was
conjugated to the P4 peptide (SEQ ID NO: 6) to form
Biotin-SKKSGSKKSGSKKSGIL called "P4-biotin." Labeling of the
N-terminus of the P4 sequence (SEQ ID NO: 6) was performed while
the peptide was still on the resin and its side chain amino acid
groups were still protected, hereinafter called P4-resin. The
P4-resin was synthesized by SynPep, lot 02GE2271, and the final
Fmoc had already been removed from the N-terminus. The coupling
reaction of biotin to peptide was achieved via standard HOBT/HBTU
chemistry, such as that used in automated peptide synthesis. The
peptide-biotin conjugate was deprotected and cleaved as described
in Example 1.
Example 12
[0057] The N-terminus of the P5 peptide (SEQ ID NO: 7) with an Ahx
linker was labeled with fluorescein in the following manner.
Labeling of the N-terminus of the P5 (SEQ ID NO: 7) with an Ahx
linker sequence was performed while the peptide was still on the
resin and its side chain amino acid groups were still protected,
hereinafter referred to as Ahx-P5-resin. The Ahx-P5-resin used in
this experiment was synthesized by SynPep, lot 027191GEN, and the
final Fmoc had already been removed from the N-terminus. The
substitution of peptide on resin was given to be 0.5 .mu.mol/mg.
Ahx-P5-resin (60 mg, 30 .mu.mol) was swollen in NMP for five
minutes in a fritted filtration vessel. Diisopropylethlamine
(DIEA), 200 .mu.L (2 M) DIEA in NMP, was added to the Ahx-P5-resin,
and then NHS-fluorescein (60 mg, 136 .mu.mol) was added under
yellow light. The mixture was protected from light, flushed gently
with nitrogen and allowed to stir for 24 hours. The solid phase was
then washed three times with NMP and three times with
dichloromethane (DCM) before being dried under nitrogen. The
peptide was cleaved and deprotected as described in Example 1.
Example 13
[0058] Peptide and subtilisin fusions were prepared using molecular
biology methods. A Bacillus subtilis strain (BS 1033, Genentech)
was obtained from Genencor International. This Bacillus strain
carried the plasmid pSS5 into which the GG36 gene construct T274A
was inserted. T274A (U.S. Pat. No. 5,185,258) was a modification of
the original Bacillus lentis (ATCC 21536) GG36 protease gene in
which the penultimate amino acid, threonine, had been converted to
an alanine with the resulting addition of a unique PstI restriction
site at this site.
[0059] As the PSS5 vector contains a PstI restriction site, T274A
was transferred to vector pBS42rending the construct amenable to
using its unique PstI site the for the insertion of peptide
sequences. The following oligonucleotides were custom made from
Operon Technologies (Alameda, Calif.):
TABLE-US-00001 R5, upper strand (SEQ ID NO: 8): GCTCGCTCCT
CCAAAAAATC CGGTTCCTAC TCCGGTTCCA AAGGTTCCAA ACGTCGTATC CTGTAATGCA
R5, bottom strand (SEQ ID NO: 9): (Seq. ID No. 9) TTACAGGATA
CGACGTTTGG AACCTTTGGA ACCGGAGTAG GAACCGGATT TTTTGGAGGA GCGAGCTGCA
R2, upper strand (SEQ ID NO: 10): (Seq. ID No. 10) GCTCGCTCCT
CCAAAAAATC CGGTTCCTAC TCCGGTTACT CCACCAAAAA ATCCGGTTCC CGTATCCTGT
AATGCA R2, bottom strand (SEQ ID NO: 11): (Seq. ID No. 11)
TTACAGGATA CGGGAACCGG ATTTTTTGGT GGAGTAACCG GAGTAGGAAC CGGATTTTTT
GGAGGAGCGA GCTGCA P4, upper strand (SEQ ID NO: 12): (Seq. ID No.
12) GCTCGCTCCA AAAAATCCGG TTCCAAAAAA TCCGGTTCCA AAAAATCCGG
TATCCTGTAA TGCA P4, bottom strand (SEQ ID NO: 13): (Seq. ID No. 13)
TTACAGGATA CCGGATTTTT TGGAACCGGA TTTTTTGGAA CCGGATTTTT TGGAGCGAGC
TGCA
[0060] The above oligo pairs are designed to be complimentary
yielding PstI "sticky ends" when annealed. Insertion of the
annealed pairs into the PstI site corresponding to the penultimate
GG36 amino acid alanine results in maintaining the alanine as well
as the final GG36 arginine. Peptide amino acid sequences are then
encoded, in frame, followed immediately by a TAA stop codon.
[0061] The above oligo pairs were mixed in equimolar amounts, 125
.mu.M each, in water. Mixtures were heated to 90.degree. C. for ten
minutes in a heating block in Hotstart (wax containing) PCR tubes.
The heating block was then switched off and allowed to cool to room
temperature over the course of .about.1 hour. 1 .mu.L of annealed
mixture was used in a ligation reaction with 1 .mu.L (ca. 250 ng)
of PstI cut, gel purified pBS42/T274A vector. Gel analysis
indicated that this resulted in an overwhelming ratio of insert to
vector. A Boeringer Mannheim "Rapid Ligation" kit was used as per
manufacturer's protocol. 5 .mu.L of each ligation mix was used to
transform competent E. Coli MM294 cells (50 .mu.l cells, mixed
thoroughly, incubated on ice 30 min, 60 second 37.degree. C. heat
shock, 2 min. on ice, 1 hour outgrowth in 150 .mu.L SOC at
37.degree. C. for 1 hour, 100 .mu.L plated to two LA-cmp5 plates).
Control plates using 1 .mu.L water in place of insert resulted in
TMTC colonies while all other plates yielded 25-30 colonies each.
Ten colonies from each different peptide insert transformation were
picked and analyzed by PCR. One of ten colonies for the R5 and R2
constructs and three of the ten P4 constructs were shown to have
the proper orientation/insertion. Correct orientation and sequences
were confirmed using DNA sequencing.
Example 14
[0062] The peptide-subtilisin fusions were expressed as proteins
using the following methods. GG36-peptide fusion plasmids were
isolated and used to transform Bacillus subtilis 3594 comK cells.
Transformants were grown on LA-cmp5 plates containing 1.6% skim
milk. Cells containing fusion plasmids as well as native GG36
(T274A) exhibited similar zones of skim milk clearing indicating
the production of active protease while untransformed cells grown
on antibiotic-free LA/skim milk plates did not. Single colonies of
the transformants were grown in 5 ml overnight tubes containing
LB-cmp5 for 9 hours at 37.degree. C. 250 rpm (OD .about.5). 50
.mu.L of this growth was used to inoculate 50 mL of FN2 Shake Flask
Medium containing 5 mg/L cmp in 250 mL fluted Erlenmeyer shake
flasks. Flasks were grown at 37.degree. C. 250 rpm. Flasks
containing native GG36 (T274A) as well as media alone were included
as controls. After 40 hrs growth, culture supernatants were
harvested by centrifugation/filtration (0.22 .mu.m) and
concentrated .about.3.times. using a Centricon device (10K MWCO).
Centricon permeate and concentrated retentates were desalted/buffer
exchanged using 25 mM tris-HCl pH 8.0 equilibrated P-10 desalting
columns (Bio-Rad).
[0063] GG36-peptide construct plasmids were transformed into
Bacillus subtilis strain AK2200 as previous. This is a strain that
has been deleted for six post-translational modification proteases
and has been used in the production of modified enzymes. Resulting
transformants demonstrated skim milk clearing, however in this case
the R5 and R2 constructs yielded smaller clearing zones than the
control GG36 (T274A) while the P4 construct yielded barely
perceptible clearing zones. Single colonies were grown in
shake-flasks and their culture supernatants processed.
Example 15
[0064] A P4 Peptide (SEQ ID NO: 6)-.beta.-lactamase (BLA) fusion
has been prepared using molecular biology methods. Plasmid pME22
containing engineered BLA was restriction digested with BbsI and
gel purified. Plasmid pME22 contains the marker that confers
chloramphenicol (cmp) resistance; properly expressed BLA confers
resistance to cefotoxime (ctx) as well. The engineered BLA also
contains a "his tag" (six histidine residues at its C-terminus) to
facilitate subsequent purification. Oligonucleotide pairs were
obtained as in Example 13.
[0065] The oligo pair was designed to be complimentary, yielding
appropriate "sticky ends" when annealed. Insertion of the annealed
pairs into the BbsI cut pME22 results in in-frame addition of
peptide DNA sequences in addition to required start signal peptide
sequences. Signal peptide is cleaved upon secretion of the fusion
protein into the cell periplasim yielding peptides fused to active
BLA. Due to the nature of BbsI cutting, pME22 cannot re-circularize
and inserts that are not properly oriented or annealed will not
result in in-frame expression of active fused BLA. The following
oligo pair was synthesized:
TABLE-US-00002 P4-BLA, upper strand (SEQ ID NO: 14): ACTAGTCGTT
CCTTTCTATT CTCACTCTTC CAAAAAATCC GGTTCCAAAA AATCCGGTTC CAAAAAATCC
GGTATCCTGA CGCCAGTGTC AGAAAAACAG CTG P4-BLA, lower strand (SEQ ID
NO: 15): CCGCCAGCTG TTTTTCTGACA CTGGCGTCA GGATACCGGA TTTTTTGGAAC
CGGATTTTTT GGAACCGGAT TTTTTGGAAG AGTGAGAATAG AAAGGAACG AC
The above oligo pair was mixed in equimolar amounts, 12.5 .mu.M
each, in water. 100 .mu.L was heated to 100.degree. C. for 2
minutes in a heating block in Hotstart (wax containing) PCR tubes.
The heating block was switched off and allowed to cool to room
temperature over the course of .about.1 hour. 2.5 .mu.L of annealed
mixture was used in a ligation reaction with 2.5 .mu.L (ca. 50 ng)
of BbsI cut, gel purified pME22 plasmid. A Takara kit (Cambrex Bio
Science Verviers S.P.R.L., BELGIUM) was used as per manufacturer's
protocol. 5 .mu.L of the 10 .mu.L ligation mix was used to
transform competent E. coli TOP10 (Invitrogen) cells (50 .mu.L
cells, mixed thoroughly, incubated on ice 30 min, 30 second
42.degree. C. heat shock, outgrowth in 250 .mu.L SOC at 37.degree.
C. for 1 hour, entire volume plated to one LA-cmp5 plates which
yielded 5-10 colonies each). Five transformants were picked and
tested for growth on LA plates containing cmp as well as ctx, 5 and
0.1 ppm respectively. All colonies grew in presence of ctx and were
analyzed by PCR. All colonies were found to contain correct plasmid
constructs by PCR; purified plasmid was used to confirm all by DNA
sequencing.
Example 16
[0066] The peptide-BLA fusion was expressed as protein using the
following methods. Single colonies of the fusion constructs as well
as a control BLA fusion (pME23) were grown in 5 mL overnight tubes
containing LB-cmp5 overnight at 37.degree. C. 250 rpm (OD
.about.5). 200 .mu.L of this growth was used to inoculate 50 mL of
TB media containing 5 mg/L cmp in 250 ml fluted Erlenmeyer shake
flasks. Flasks were grown at 37.degree. C. 250 rpm. After 24 hrs
growth, culture supernatants were harvested by
centrifugation/filtration (0.22 .mu.m) and cell pellets were stored
at -20.degree. C.
[0067] Supernatants were concentrated .about.3.times. using a
Centricon device (10K MWCO). Centricon permeate and concentrated
retentates were desalted/buffer exchanged using 25 mM tris-HCl pH
8.0 equilibrated P-10 desalting columns (Bio-Rad).
[0068] Periplasmic fusion protein was purified using a Pro-Bond kit
(Invitrogen) optimized for the affinity purification of "his
tagged" proteins as per manufacturer's protocol. Concentrated
supernatants and Pro-Bond purified material was analyzed by
SDS-PAGE (NuPage gels, 4-12%, MES buffer). Fusion protein appears
to have its expected molecular weight as determined by MALDI-TOF
mass spectrometry. N-terminal protein sequencing by Edman
Degradation confirms that the fusion is mostly intact, the P4 (SEQ
ID NO:6) moiety being truncated by two amino acids.
Example 17
[0069] A nanocomposite utilizing the R5-fluorescein peptide
conjugate was formed. A silicic acid solution was formed by
dissolving 0.208 g (1 M) tetraethylorthosilicate
(tetraethoxysilane, TEOS) in 1 mM HCl in deionized water (1 mL
total) for 6-18 hours. 100 .mu.L (1 M) silicic acid solution was
added to 1 mg/mL fluorescein-R5 peptide (SEQ ID NO 1) conjugate in
900 .mu.L (25 mM Tris-HCl) buffer, pH 8. The reaction was allowed
to run for half an hour on a rotary mixer. The reaction mixture was
then centrifuged at 14,000 rpm to spin down precipitate. The
solution was removed with a pipette and the remaining material was
mixed with deionized water and centrifuged again. Precipitate was
washed at least twice in this manner, frozen at -80.degree. C., and
lyophilized. The composite was fluorescent under ultraviolet light
and possessed a different morphology than the composite derived
from the unlabelled R5 peptide as imaged by SEM.
Example 18
[0070] A nanocomposite was formed from combination of a T-unit
silane with fluorescein labeled P5 peptide (SEQ ID NO 7),
hereinafter referred to as P5-fluorescein. A solution of 241 .mu.L
phenyltriethoxysilane (PhSi(OEt).sub.3), 234.5 .mu.L (60 mM) HCl
(aq.), and 296 .mu.L ethanol was allowed to react for 2 hours,
after which phenyltriethoxysilane was considered hydrolyzed. First
100 .mu.L of P5-fluorescein (10 mg/mL in deionized water) was added
to 800 .mu.L Tris-HCl (25 mM) buffer, followed by 100 .mu.L
pre-hydrolyzed phenyltriethoxysilane solution. The reaction was
performed in triplicate and the solutions were allowed to stir 10
minutes; the precipitated material was an orange color indicating
the presence the P5-fluorescein peptide. The reactions were
centrifuged at 14,000 rpm for 15 minutes, re-suspended in purified
water, centrifuged again, and the pellet remaining was lyophilized.
The presence of the P5-fluorescein peptide in the composite was
further confirmed by mass spectrometry.
Example 19
[0071] Peptides of the present invention were found to produce a
novel product when exposed to T-unit silanes. 23.4 .mu.L (0.1 M)
3-aminopropyltriethoxysilane or 24.1 .mu.L (0.1 M)
phenyltriethoxysilane was added directly to a 10 mM Tris-HCl
buffered R5 peptide (SEQ ID NO: 1) solution (1.5-1.9 mg/mL) at
either pH 7 or pH 8 for a total volume of 1 mL. The assays were
allowed to run overnight on a rotary mixer. The samples, including
experimental controls that lacked peptide, appeared foamy and could
not be centrifuged at 14,000 rpm. All samples were frozen at
-80.degree. C. and lyophilized. Selected samples were then analyzed
by SEM imaging and SEM-EDS analysis. Imaging of the precipitated
material by SEM showed clear differences in morphology between
control (no peptide) and experimental preparations. Whereas the
control preparations were completely amorphous, the
peptide-precipitated material contained square-shaped features on
the order of 500 to 1000 nm.
Example 20
[0072] A slow-addition reaction to promote the monodispersity of
nanoparticles is performed. A solution of 0.1 M silicic acid is
made by dissolving 20.8 mg TEOS in 1 mM HCl for a total volume of 1
mL. This silicic acid solution is added incrementally to a peptide
solution of 1.1 mg R5 (SEQ ID NO: 1) in 800 .mu.L (25 mM) Tris-HCl
buffer, pH 8. Aliquots of 10 .mu.L each of the silicic acid
solution are added every 30 seconds for 10 minutes, resulting in a
total reaction volume of 1 mL at the end of the slow addition
processes. The reaction mixture is then centrifuged at 14,000 rpm
to spin down precipitate. The solution is removed with a pipette
and the remaining material is mixed with deionized water and
centrifuged again. The precipitate is washed at twice in this
manner, frozen at -80.degree. C., and lyophilized.
Example 21
[0073] Nanocomposites were precipitated using a number of peptides
and peptide derivatives as shown in Table 1 in accordance with the
following procedure. 0.208 g (1 M) tetraethylorthosilicate (TEOS)
was first dissolved in 1 mM HCl in deionized filtered water (1 mL
total) for 6-18 hours to make a silicic acid solution. The assay
contained 100 .mu.L (1 M) silicic acid solution added to 100 .mu.L
(10 mg/mL) peptide in 800 .mu.L (50 mM) sodium borate buffer, pH
8.5. The reaction was generally allowed to run for half an hour or
more on a rotary mixer. The reaction mixture and controls (with
unmodified peptide and without peptide) were then centrifuged at
14,000 rpm to spin down any precipitate. The supernatant was
removed with a pipette and the remaining material was mixed with
deionized water and centrifuged again. Precipitate was washed at
least twice in this manner, frozen at .sup.-80.degree. C., and
lyophilized. The reactions were performed in duplicate. The mass of
the lyophilized material recovered from each experiment is given
below:
TABLE-US-00003 TABLE 1 Sample 1 Sample 2 Name (+silane) (+/-0.2 mg)
(+/-0.2 mg) P4 (SEQ ID NO: 6) 0.5 mg 0.8 mg P4-C12 0.4 mg 0.5 mg
P4-cholest 0.4 mg 0.3 mg P4-EDTA less than 0.2 mg less than 0.2 mg
No peptide None observed None observed
Example 22
[0074] Silica precipitation using a biotinylated P4 peptide (SEQ ID
NO: 6) was performed. A 10 mg/mL solution of P4-biotin was made in
deionized water. This peptide solution (100 .mu.L, 1 mg/mL final
concentration) was added to borate buffer (800 .mu.L, 50 mM) at pH
8.5. Silicic acid, made from 1 M TEOS in 1 mM aqueous HCl stirred
overnight, was added (100 .mu.L) to the buffered peptide. A very
fine precipitate was observed within the first two minutes of
reaction time. The final solution, at a pH of 8.0+/-0.2, was
allowed to stir at room temperature for 10 minutes before the first
centrifugation. The 1 mL aliquot was spun on an ultracentrifuge for
12-15 min. at 14,000 g. The liquid was removed and the precipitate
was resuspended in deionized water. The precipitate was then spun
and washed twice more with deionized water.
Example 23
[0075] Q/T-unit mixed-resin composites were formed with
silica-precipitating peptides as shown in Table 2. 29 .mu.L
(.about.0.02 M) methyltrimethoxysilane and 184 .mu.L (0.08 M)
tetraethylorthosilicate were dissolved in 1 mM HCl in deionized
filtered water (1 mL total) for 6-18 hours to make a homogenous
solution of mixed Q/T prehydrolyzed solution. 100 .mu.L (1 M) of
the mixed Q/T prehydrolyzed solution added to 100 .mu.L (10 mg/mL)
peptide in 800 .mu.L (50 mM) sodium borate buffer, pH 8.5. The
reactions were allowed to run 10 minutes on a rotary mixer. The
reaction mixture and controls (with unmodified peptide and without
peptide) were then centrifuged at 14,000 rpm to spin down any
precipitate. The supernatant was removed with a pipette and the
remaining material was mixed with deionized water and centrifuged
again. Precipitate was washed at least twice in this manner, frozen
at .sup.-80.degree. C., and lyophilized.
[0076] The mass of the lyophilized material recovered from each
experiment is given below:
TABLE-US-00004 TABLE 2 Sample 1 Name (+Q/T) (+/-0.2 mg) P4 (SEQ ID
NO: 6) 0.3 mg P4-C12 2.2 mg P4-cholesterol 0.7 mg P4-EDTA less than
0.2 mg No peptide None observed
Example 24
[0077] The ability to modify a surface using the peptide
derivatives of the present invention was confirmed. Glass
microscope slides were cleaned by treatment with a solution of
ethanolic KOH (3 M) for 10 minutes followed by sequential washing
with 1 M Tris-HCl, pH 8 and deionized water. The labeled peptides
R5-fluorescein and R5-pyrene were applied to the treated glass
surface in two ways. In the first method, the glass slide was dip
coated in a peptide solution (10 mg/mL). The second method used a
solution of peptide (10 mg/mL) in ethanol, which was applied
manually in several layers, allowing the ethanol to evaporate
between layers. In both cases, the presence of the peptide film was
visually confirmed by examining the glass slides under UV
light.
[0078] A silicic acid solution as described in Example 13 was then
spotted onto the peptide film with a pipette tip. After five
minutes, the slides were washed several times by vigorous agitation
in deionized water. The spots containing the peptide-silica
nanocomposite adhered to the glass slide and showed fluorescence
under UV light, whereas the unreacted peptide film was no longer
present. Controls using buffer (25 mM Tris-HCl, pH 8) instead of
silicic acid solution did not result in peptide retention on the
surface following the final wash. To demonstrate the potential for
surface patterning with this technique, the silicic acid solution
was applied to the peptide film in a series of dots resulting in
the formation of a peptide/silica composite array.
Example 25
[0079] The P5-fluorescein-silica nanocomposite as made in Example
18 was used to label Daudi cells. Aliquots (1-2 mg) of both the
P5-fluorescein peptide and the P5-fluorescein-silica nanocomposite
were resuspended in PBS buffer (1.2 ml) containing 0.05% bovine
serum albumin (BSA) and centrifuged to remove unsuspended solids.
Daudi Cells (7.times.10.sup.7 total), obtained from the ATTC
(Manassas, Va.) and cultured under recommended conditions, were
mixed with the peptide ligands and incubated for 2.5 hr at
37.degree. C. in 3 ml of PBS/BSA buffer. The total fluorescence of
the solutions was measured and expressed in terms of relative
fluorescence units (RFU). Controls for ligands with no cells, and
cells with no ligand were run in parallel. Duplicate tests were run
for cells with ligands and single tests were run for others. The
RFU levels of the control samples with ligand alone were subtracted
from the ligand plus cell samples. Following incubation, cells were
washed twice in 10 ml PBS/BSA buffer, resuspended in 2.6 ml buffer
and two aliquots of 0.2 ml were assayed. Fluorescence measurements
of the cell fraction indicated that the P5-fluorescein peptide
alone bound poorly to the cells (0.16 RFU, 0.07% of total RFU
added), whereas the P5-fluorescein-silica nanocomposite bound
14-fold more efficiently (7.8 RFU, 1% of total RFU).
Example 26
[0080] A silica nanocomposite was synthesized with pyrene labeled
peptides mediated by an electric field. The R5-pyrene peptide as
formed in example 8 was contacted with a silicate solution in 50 mM
borate buffer, pH 8.0 in a 0.5% agarose matrix under the influence
of an electric field as follows. Standard gel electrophoresis
equipment was used and the gel matrix was 0.5% agarose. A small
well (50-100 mm.sup.2) was cut into the agarose matrix in the
experimental lane near the negative electrode and filled with a 1 M
sodium silicate solution, pH 8.5 which had been freshly prepared by
dilution of a 6.25 M stock solution with deionized water and pH
adjustment with Amberlite IRA-118, H.sup.+ resin. A corresponding
well in the control lane contained 50 mM sodium borate buffer, pH
8.0. Similar wells nearest to the positive electrode contained 200
.mu.L each of the R5-pyrene peptide (10 mg/mL). A middle well in
each lane contained 50 mM sodium borate buffer, pH 8.0. A potential
(120 V) was applied across the electrodes and the peptide bands
(control and experimental) were observed under UV light to move
through the gel toward the negative electrode. The peptide in the
control lane moved continuously in a narrow band. The peptide in
the experimental lane was arrested and then appeared to spread out,
after which no movement was observed in the experimental lane.
Observation of the experimental lane under a fluorescence
microscope revealed the formation of dispersed fluorescent
particles embedded within the agarose matrix, the size of which
were estimated to be in the 100-200 nm range. Such particles were
not observed in the control lane.
Sequence CWU 1
1
26119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys
Gly Ser Lys Arg1 5 10 15Arg Ile Leu221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Ser
Ser Lys Lys Ser Gly Ser Tyr Ser Gly Tyr Ser Thr Lys Lys Ser1 5 10
15Gly Ser Arg Ile Leu20322PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Leu Asp Ala Gln Glu Arg Arg
Arg Glu Arg Arg Ala Glu Lys Gln Glu1 5 10 15Gln Trp Lys Ala Ala
Asn20419PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ser Ser His Lys Ser Gly Ser Tyr Ser Gly Ser His
Gly Ser His Arg1 5 10 15Arg Ile Leu519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Cys
Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys Gly Ser Lys Arg1 5 10
15Arg Cys Leu617PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 6Ser Lys Lys Ser Gly Ser Lys Lys Ser Gly
Ser Lys Lys Ser Gly Ile1 5 10 15Leu79PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Arg
Arg Arg Arg Arg Arg Arg Arg Arg1 5870DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gctcgctcct ccaaaaaatc cggttcctac tccggttcca
aaggttccaa acgtcgtatc 60ctgtaatgca 70970DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9ttacaggata cgacgtttgg aacctttgga accggagtag
gaaccggatt ttttggagga 60gcgagctgca 701076DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gctcgctcct ccaaaaaatc cggttcctac tccggttact
ccaccaaaaa atccggttcc 60cgtatcctgt aatgca 761176DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11ttacaggata cgggaaccgg attttttggt ggagtaaccg
gagtaggaac cggatttttt 60ggaggagcga gctgca 761264DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12gctcgctcca aaaaatccgg ttccaaaaaa tccggttcca
aaaaatccgg tatcctgtaa 60tgca 641364DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13ttacaggata ccggattttt tggaaccgga ttttttggaa
ccggattttt tggagcgagc 60tgca 6414103DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14actagtcgtt cctttctatt ctcactcttc caaaaaatcc
ggttccaaaa aatccggttc 60caaaaaatcc ggtatcctga cgccagtgtc agaaaaacag
ctg 10315103DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 15ccgccagctg tttttctgac
actggcgtca ggataccgga ttttttggaa ccggattttt 60tggaaccgga ttttttggaa
gagtgagaat agaaaggaac gac 1031633PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 16Ser Ser Lys Lys Ser Gly
Ser Tyr Tyr Ser Tyr Gly Thr Lys Lys Ser1 5 10 15Gly Ser Tyr Ser Gly
Tyr Ser Thr Lys Lys Ser Ala Ser Arg Arg Ile20 25
30Leu1719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys
Gly Ser Lys Arg1 5 10 15Arg Asn Leu1812PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Ala
Pro Pro Gly His His His Trp His Ile His His1 5 101912PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Met
Ser Ala Ser Ser Tyr Ala Ser Phe Ser Trp Ser1 5 102012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Lys
Pro Ser His His His His His Thr Gly Ala Asn1 5 102112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Met
Ser Pro His Pro His Pro Arg His His His Thr1 5 102212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Met
Ser Pro His His Met His His Ser His Gly His1 5 102312PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Leu
Pro His His His His Leu His Thr Lys Leu Pro1 5 102412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Ala
Pro His His His His Pro His His Leu Ser Arg1 5 102512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Arg
Gly Arg Arg Arg Arg Leu Ser Cys Arg Leu Leu1 5 1026265PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Met
Lys Leu Thr Ala Ile Phe Pro Leu Leu Phe Thr Ala Val Gly Tyr1 5 10
15Cys Ala Ala Gln Ser Ile Ala Asp Leu Ala Ala Ala Asn Leu Ser Thr20
25 30Glu Asp Ser Lys Ser Ala Gln Leu Ile Ser Ala Asp Ser Ser Asp
Asp35 40 45Ala Ser Asp Ser Ser Val Glu Ser Val Asp Ala Ala Ser Ser
Asp Val50 55 60Ser Gly Ser Ser Val Glu Ser Val Asp Val Ser Gly Ser
Ser Leu Glu65 70 75 80Ser Val Asp Val Ser Gly Ser Ser Leu Glu Ser
Val Asp Asp Ser Ser85 90 95Glu Asp Ser Glu Glu Glu Glu Leu Arg Ile
Leu Ser Ser Lys Lys Ser100 105 110Gly Ser Tyr Tyr Ser Tyr Gly Thr
Lys Lys Ser Gly Ser Tyr Ser Gly115 120 125Tyr Ser Thr Lys Lys Ser
Ala Ser Arg Arg Ile Leu Ser Ser Lys Lys130 135 140Ser Gly Ser Tyr
Ser Gly Tyr Ser Thr Lys Lys Ser Gly Ser Arg Arg145 150 155 160Ile
Leu Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly Ser Lys Gly Ser165 170
175Lys Arg Arg Ile Leu Ser Ser Lys Lys Ser Gly Ser Tyr Ser Gly
Ser180 185 190Lys Gly Ser Lys Arg Arg Asn Leu Ser Ser Lys Lys Ser
Gly Ser Tyr195 200 205Ser Gly Ser Lys Gly Ser Lys Arg Arg Ile Leu
Ser Ser Lys Lys Ser210 215 220Gly Ser Tyr Ser Gly Ser Lys Gly Ser
Lys Arg Arg Asn Leu Ser Ser225 230 235 240Lys Lys Ser Gly Ser Tyr
Ser Gly Ser Lys Gly Ser Lys Arg Arg Ile245 250 255Leu Ser Gly Gly
Leu Arg Gly Ser Met260 265
* * * * *