U.S. patent application number 11/352782 was filed with the patent office on 2007-05-24 for carbon nanotube binding peptides.
Invention is credited to Anand Jagota, Steven Raymond Lustig, Hong Wang, Siqun Wang.
Application Number | 20070117149 11/352782 |
Document ID | / |
Family ID | 29715378 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117149 |
Kind Code |
A1 |
Jagota; Anand ; et
al. |
May 24, 2007 |
Carbon nanotube binding peptides
Abstract
Peptides have been generated that have binding affinity to
carbon nanostructures and particularly carbon nanotubes. Peptides
of or the invention are generally about twelve amino acids in
length. Methods for generating carbon nanotube binding peptides are
also disclosed.
Inventors: |
Jagota; Anand; (Bethlehem,
PA) ; Lustig; Steven Raymond; (Landenberg, PA)
; Wang; Siqun; (Wilmington, DE) ; Wang; Hong;
(Kennett Square, 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: |
29715378 |
Appl. No.: |
11/352782 |
Filed: |
February 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10453415 |
Jun 3, 2003 |
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11352782 |
Feb 13, 2006 |
|
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60413273 |
Sep 25, 2002 |
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60385696 |
Jun 4, 2002 |
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Current U.S.
Class: |
435/7.1 ;
530/350; 977/746 |
Current CPC
Class: |
C07K 7/08 20130101; C07K
1/047 20130101; Y10S 977/742 20130101; B82Y 30/00 20130101; A61K
47/6925 20170801; B82Y 5/00 20130101; Y10S 977/705 20130101; C07K
17/02 20130101 |
Class at
Publication: |
435/007.1 ;
530/350; 977/746 |
International
Class: |
C40B 40/10 20060101
C40B040/10; C07K 14/705 20060101 C07K014/705 |
Claims
1-7. (canceled)
8. A method of immobilizing a carbon nanotube comprising: a)
immobilizing a carbon nanotube binding peptide having the general
structure: N-M-C wherein: N is the N-terminal portion of the
peptide having about 4 amino acids, 75% of which are hydrophilic; M
is the median portion of the peptide having about 4 amino acids,
75% of which are hydrophobic; and C is the C-terminal portion of
the peptide having about 4 amino acids 75% of which are
hydrophilic; and b) contacting a carbon nanostructure with the
immobilized peptide of (a) whereby the nanostructure is
immobilized.
9. A method according to claim 8 wherein the carbon nanotube
binding peptide has an amino acid sequence selected from the group
consisting of SEQ ID NOs:1-24, SEQ ID NOs:35-39, SEQ ID NOs:40-85,
SEQ ID NOs:86-113, SEQ ID NOs:114-147, and SEQ ID NOs:148-177 or an
amino acid sequence selected from the group consisting of SEQ ID
NOs:1-24, SEQ ID NOs:35-39, SEQ ID NOs:40-85, SEQ ID NOs:86-113,
SEQ ID NOs:114-147, and SEQ ID NOs:148-177 wherein the sequence
contains at least one amino acid substitution with a chemically
equivalent amino acid.
10-22. (canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application 60/413,273 filed on Sep. 25, 2002 and U.S. Application
60/385,696 filed on Jun. 4, 2002.
FIELD OF THE INVENTION
[0002] The invention relates to methods, and compositions useful
for manipulation, purification and characterization of carbon
nanotubes. More specifically, the invention relates to peptides
that bind carbon based nanostructures, their synthesis and methods
of use.
BACKGROUND OF THE INVENTION
[0003] Carbon nanotubes (CNT) have been the subject of intense
research since their discovery in 1991. CNT's possess unique
properties such as small size and electrical conductivity, which
makes them suitable in a wide range of applications, including use
as structural materials in molecular electronics, nanoelectronic
components, and field emission displays. Carbon nanotubes may be
either multi-walled (MWNTs) or single-walled (SWNTs), and have
diameters in the nanometer range. Depending on their atomic
structure CNT's may have either metallic or semiconductor
properties, and these properties, in combination with their small
dimensions makes them particularly attractive for use in
fabrication of nano-devices.
[0004] One of the drawbacks to the implementation of CNT's in
nano-device fabrication processes is the difficulty in obtaining
samples of CNT's that have uniform lengths, or chirality.
Additionally, no facile method is available for the immobilization
and manipulation of CNT's for nano-device fabrication.
[0005] Most methods of CNT synthesis produce a product that is a
mixture of entangled tubes of "ropes", giving CNT's differing in
diameter, chirality, and in the number of walls. Various methods
such as acid washing, ultra-sonification, polymer wrapping and use
of surfactants have been employed for nanotube separation (J. Liu
et al. Science 280, 1253 (1998); A. G. Rinzler, Appl. Phys. 67, 29
(1998); A. C. Dillion et al. Adv. Mater. 11, 1354 (1999);
(Schlittler et al. Science 292:1136 (2001)).). However, there has
been no report of a method for the specific disentangling of
nanotube ropes or their separation into populations having discrete
sizes, chirality or conducting properties.
[0006] Because of their ability to specifically recognize
substrates, various proteins represent one possible route to
solving the CNT separation/purification problem as well as
providing a possible means for CNT immobilization. Some attempts
have been made to raise antibodies to various carbon based
structures. For example, Chen et al. (WO 01/16155 A1) used
conjugated fullerenes to raise monoclonal antibodies to C.sub.60
fullerene as a hapten. However, the population of antibodies raised
by immunization of mice with this C.sub.60 fullerene derivative
which was conjugated to bovine thyroglobulin included a
sub-population that cross reacted with a C.sub.70 fullerene. No
attempts have been made to date to raise antibodies to carbon based
nanotubes.
[0007] Since its introduction in 1985 phage display has been widely
used to discover a variety of ligands including peptides, proteins
and small molecules for drug targets. (Dixit, S., J. of Sci. &
Ind. Research, 57, 173-183, 1998). The applications have expanded
to other areas such as studying protein folding, novel catalytic
activities and DNA-binding proteins with novel specificities.
Whaley et al (Nature, 405:665 (2000)) has used phage display
technique to identify peptide sequences that can bind specifically
to different crystallographic forms of inorganic semiconductor
substrates. Although the method of generating large, diverse
peptide libraries with phage display has been known for some time,
it has not been applied to the problem of finding peptides that may
be useful in the binding and manipulation of CNT's.
[0008] The problem to be solved, therefore, is to provide materials
that have binding specificity to CNT's and other carbon based
nanostructures so that they may be used in separation and
immobilization of these structures for the fabrication of
nano-devices. Applicants have solved the stated problem by
providing a series of carbon nanotube binding peptides with high
affinity and specificity for CNT's.
SUMMARY OF THE INVENTION
[0009] In one aspect the invention provides a process for
generating a carbon nanostructure binding peptide comprising:
[0010] a) providing a library of randomly generated peptides;
[0011] b) providing a sample of a carbon nanostructure; [0012] c)
contacting the library of (a) with the carbon nanostructure of (b)
whereby a subset of the peptide library of (a) binds to said
nanostructure to create a first peptide sub-library; [0013] d)
screening the first peptide sub-library of (c) for the presence of
multiples of the same sequence wherein the existence of at least
one multiple of a sequence indicates a carbon nanostructure binding
peptide.
[0014] Preferred methods of generating the peptides of the
invention include phage display, bacterial display, yeast display
and combinatorial solid phase peptide synthesis.
[0015] The invention additionally provides a carbon nanotube
binding peptide having an amino acid sequence selected from the
group consisting of SEQ ID NOs:1-24, SEQ ID NOs:35-39, SEQ ID
NOs:40-85, SEQ ID NOs:86-113, SEQ ID NOs:114-147, and SEQ ID
NOs:148-177 or an amino acid sequence selected from the group
consisting of SEQ ID NOs:1-24, SEQ ID NOs:35-39, SEQ ID NOs:40-85,
SEQ ID NOs:86-113, SEQ ID NOs:114-147, and SEQ ID NOs:148-177
wherein the sequence contains at least one amino acid substitution
with a chemically equivalent amino acid.
[0016] In another embodiment the invention provides a method of
immobilizing a carbon nanotube comprising: [0017] a) immobilizing a
carbon nanotube binding peptide having the general structure: N-M-C
[0018] wherein: [0019] N is the N-terminal portion of the peptide
having about 4 amino acids, 75% of which are hydrophilic; [0020] M
is the median portion of the peptide having about 4 amino acids,
75% of which are hydrophobic; and [0021] C is the C-terminal
portion of the peptide having about 4 amino acids 75% of which are
hydrophilic; and [0022] b) contacting a carbon nanostructure with
the immobilized peptide of (a) whereby the nanostructure is
immobilized.
[0023] In another embodiment the invention provides a method of
dispersing a population of carbon nanotube ropes comprising: [0024]
a) providing a population of carbon nanotubes in solution in rope
formation; and [0025] b) contacting the population of carbon
nanotubes of step (a) with a carbon nanotube binding peptide having
the general structure: N-M-C [0026] wherein: [0027] N is the
N-terminal portion of the peptide having about 4 amino acids, 75%
of which are hydrophilic; [0028] M is the median portion of the
peptide having about 4 amino acids, 75% of which are hydrophobic;
and [0029] C is the C-terminal portion of the peptide having about
4 amino acids 75% of which are hydrophilic; [0030] whereby the
carbon nanotube ropes are dispersed.
[0031] Additionally the invention provides a process for generating
a carbon nanostructure binding peptide comprising: [0032] a)
providing a library of phages expressing peptides in solution;
[0033] b) providing a population of carbon nanostructures; [0034]
c) contacting the phage of (a) with the nanostructures of (b) for a
time sufficient to permit binding of the phage to the
nanostructures and form a phage-nanostructure complex; [0035] d)
removing unbound phage; [0036] e) contacting the
phage-nanostructure complex of (c) with a suitable bacterial host
whereby the bacteria are infected by the phage; [0037] f) growing
the infected bacteria of step (e) for a time sufficient to permit
replication of the phage and the expressed peptide; and [0038] g)
isolating the replicated phage and expressed peptide of step (f)
wherein the peptide binds carbon nanostructures.
[0039] Also provided herein are methods for assembling carbon
nanotubes comprising contacting a solid substrate coated with at
least one species of carbon nanotube binding peptide with a
population of carbon nanotubes whereby the carbon nanotubes bind to
the coated substrate and are assembled.
[0040] Additionally useful in the present invention are non-CNT
binding peptides having the amino acid sequence selected from the
group consisting of SEQ ID NO:28 and SEQ ID NO:34.
[0041] Provided herein are also compositions comprising a solid
substrate coated with a carbon nanotube binding peptide, as well as
compositions comprising a solid substrate coated with a carbon
nanotube binding peptide having at least one carbon nanotube bound
thereto.
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTINGS
[0042] FIG. 1 is a TEM image of phages with carbon nanotube binding
peptides on surface of carbon nanotubes.
[0043] FIG. 2A is an electronmicrograph of untreated nanotubes
ropes.
[0044] FIG. 2B is an electronmicrograph of single walled nanotubes
treated with carbon nanotube binding peptide as set forth in SEQ ID
NO:13.
[0045] FIG. 2C is an electronmicrograph of single walled nanotubes
treated with a control peptide, having little or no binding
affinity for CNT's.
[0046] FIG. 3A is an electronmicrograph of a microsphere coated
with a non-CNT binding control phage after exposure to SWNT.
[0047] FIG. 3B is an electronmicrograph of a microsphere coated
with a CNT-binding phage after exposure to SWNT.
[0048] FIG. 3C is an electronmicrograph of a microsphere coated
with a non-CNT binding peptide after exposure to SWNT.
[0049] FIG. 3D is an electronmicrograph of a microsphere coated
with a CNT binding peptide after exposure to SWNT.
[0050] 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 37 C.F.R. .sctn.1.822.
[0051] SEQ ID Nos:1-24 and 35-39 are carbon nanotube binding
peptides of the invention.
[0052] SEQ ID NOs:25, and 32 are derivatized carbon nanotube
binding peptides having a polyglycine tail.
[0053] SEQ ID Nos:26 and 27 are mutant carbon nanotube binding
peptides having a serine substituted in place of a tryptophan at
position 6.
[0054] SEQ ID NO:28 is a control peptide that have little or no
binding affinity for carbon nanostructures.
[0055] SEQ ID NO:29 is a charged portion of a carbon nanotube
binding peptide.
[0056] SEQ ID NO:30 is a polar portion of a carbon nanotube binding
peptide.
[0057] SEQ ID NO:31 is a hydrophobic portion of a carbon nanotube
binding peptide.
[0058] SEQ ID NO:33 is a primer used for sequencing M13 phage.
[0059] SEQ ID NO:34 is a non-CNT binding peptide.
[0060] SEQ ID NOs:40-85 are peptides raised against and binding to
single walled nanotoubes.
[0061] SEQ ID NOs:86-147 and 177 are peptides raised against and
binding to multiwalled carbon nanotubes.
[0062] SEQ ID NOs:148-176 are peptides raised against and binding
to graphite cleaned carbon nanotubes.
DETAILED DESCRIPTION OF THE INVENTION
[0063] The present invention provides various carbon nanotube
binding peptides generated by the process of peptide phage display.
The peptides are useful for the manipulation of carbon based
nanostructures in the fabrication of nano-devices as well as in the
separation and purification of nanotubes from mixed CNT
populations.
[0064] The peptides of the invention are particularly useful as
ligands for the assembly of carbon nanotubes and related molecules
into conducting nano devices for use in electronic applications
such as field-emission transistors, artificial actuators,
molecular-filtration membranes, energy-absorbing materials,
molecular transistors, and other optoelectronic devices as well as
in gas storage, single-electron devices, and chemical and
biological sensors.
[0065] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0066] "CNBP" means Carbon nanotube binding peptide
[0067] "HRTEM" means high-resolution transmission electron
microscopy
[0068] "MWNT" means Multi-walled nanotube
[0069] "SWNT" means Single walled nanotube
[0070] "PEG" means polyethylene glycol
[0071] "pfu" means plaque forming units
[0072] "TEM" means transmission electron microscopy
[0073] "CNT" means carbon nanotube
[0074] The term "peptide" refers to two or more amino acids joined
to each other by peptide bonds or modified peptide bonds. Peptides
include those modified either by natural processes, such as
processing and other post-translational modifications, but also
chemical modification techniques. The modifications can occur
anywhere in a peptide, including the peptide backbone, the amino
acid side chain, and the amino or carboxyl terminal. Examples of
modifications include but are not limited to amidation, acylation,
acetylation, cross linking, cyclization, glycosylation,
hydroxylation, phosphorylation, racemization, and covalent
attachment of various moieties such as nucleotide or nucleotide
derivative, lipid or lipid derivatives (see, for instance,
Proteins--Structure and Molecular Properties, 2.sup.nd Ed
Creighton, W.H. Freeman and Company, New York (1993) and
Post-translation covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York (1983)).
[0075] As used herein, the term "peptide" and "polypeptide" will be
used interchangeably.
[0076] The term "nanotube" refers to a hollow article having a
narrow dimension (diameter) of about 1-200 nm and a long dimension
(length), where the ratio of the long dimension to the narrow
dimension, i.e., the aspect ratio, is at least 5. In general, the
aspect ratio is between 10 and 2000.
[0077] By "carbon-based nanotubes" or "carbon nanotube" herein is
meant hollow structures composed primarily of carbon atoms. The
carbon nanotube can be doped with other elements, e.g., metals.
[0078] The term "carbon nanotube product" refers to cylindrical
structures made of rolled-up graphene sheet, either single-wall
carbon nanotubes or multi-wall carbon nanotubes.
[0079] The term "carbon nanotube rope" means a population of
non-aligned nanotubes.
[0080] The term "carbon nanostructure binding peptide" refers to
peptides that were selected to bind with a carbon nanostructures.
Where peptides are generated with specific affinity to carbon
nanotubes, these peptides will be referred to as carbon nanotube
binding peptides or CNBP's.
[0081] The term "stringency" as it is applied to the selection of
CNBP's means the concentration of eluting agent (usually detergent)
used to elute peptides from CNT's.
[0082] The term "peptide-nanotube complex" means structure
comprising a peptide bound to a nanotube via a binding site on the
peptide.
[0083] The term "nano-structure" means tubes, rods, cylinders,
bundles, wafers, disks, sheets, plates, planes, cones, slivers,
granules, ellipsoids, wedges, polymeric fibers, natural fibers, and
other such objects which have at least one characteristic dimension
less than about 100 nm.
[0084] The term "solid substrate" means a material to which a
carbon nanotube or binding peptide may be affixed either by direct
chemical means or via an intermediate material such as a
coating.
[0085] The term "identity" refers to a relationship between two or
more polynucleotide sequences or two or more polypeptide sequences,
as determined by comparing the sequences. "Identity" and
"similarity" can be readily calculated by known methods including
but not limited to those described in (Sequence Analysis in
Molecular Biology, Von Heinje, G., Academic Press, 1987, Sequence
analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991, and Computer Analysis of Sequence Data, Part
1, Griffin, A. M., and Griffin, H. G., eds, Humana Press, New
Jersey, 1994.
[0086] The term "amino acid" will refer to the basic chemical
structural unit of a protein or polypeptide. The following
abbreviations will be 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 Asparagine or aspartic acid Asx B Cysteine Cys C
Glutamine Gln Q Glutamine acid Glu E Glutamine or glutamic acid Glx
Z Glycine Gly G Histidine His H 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
[0087] The term "variant(s)" refers to a polynucleotide, or
polypeptide, that differs from a reference polynucleotide, or
polypeptide, respectively, but retains essential properties.
Changes in the nucleotide sequence of the variant may or may not
alter the amino acid sequence of polypeptide encoded by the
reference polynucleotide. Nucleotide changes may result in amino
acid substitutions, deletions, additions, fusions, and truncations
in the polypeptide encoded by the reference sequence. A typical
variant of a polypeptide may differ in amino acid sequence from
another reference polypeptide by one or more substitutions,
additions, deletions in any combinations. A substituted or inserted
amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a
naturally occurring such as allelic variant, or may not be known as
naturally occurring variant. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by direct synthesis,
mutagenesis techniques, or by other recombinant methods known in
the art.
[0088] The term "chemically equivalent amino acid" will refer to an
amino acid that may be substituted for another in a given protein
without altering the chemical or functional nature of that protein.
For example, it is well known in the art that alterations in a gene
which result in the production of a chemically equivalent amino
acid at a given site, but do not effect the functional properties
of the encoded protein are common. For the purposes of the present
invention substitutions are defined as exchanges within one of the
following five groups:
Hydrophobic
[0089] Small aliphatic, nonpolar or slightly polar residues: Ala,
Ser, Thr Pro, Gly; [0090] Large aliphatic, nonpolar residues: Met,
Leu, Ile, Val Cys; and [0091] Large aromatic residues: Phe, Tyr,
Trp; Hydrophilic: [0092] Polar, negatively charged residues and
their amides: Asp, Asn, Glu, Gln; [0093] Polar, positively charged
residues: His, Arg, Lys; Thus, alanine, a hydrophobic amino acid,
may be substituted by another less hydrophobic residue (such as
glycine) or a more hydrophobic residue (such as valine, leucine, or
isoleucine). Similarly, changes which result in substitution of one
negatively charged residue for another (such as aspartic acid for
glutamic acid) or one positively charged residue for another (such
as lysine for arginine) can also be expected to produce a
functionally equivalent product. Additionally, in many cases,
alterations of the N-terminal and C-terminal portions of the
protein molecule would also not be expected to alter the activity
of the protein.
[0094] "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.
[0095] "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.
[0096] "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 site, effector binding site and stem-loop structure.
[0097] "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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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 bacteriophages are derived from
two "wild" phages, called M13 and lambda. Lambda phages are used to
clone segments of DNA in the range of around 10-20 kb. They are
lytic phages. i.e., they replicate by lysing their host cell and
releasing more phages. 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.
[0103] 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 could 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.
[0104] Standard recombinant DNA and molecular cloning techniques
used here 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).
[0105] The present invention provides peptides that bind carbon
nanostructures as well as methods for generating the same and uses
thereof.
Carbon Nanostructures
[0106] The invention relates to the generation of peptides with
binding affinities for carbon nanostuctures, and particularly
nanotubes. Carbon nano-structures of the present invention are
those structures comprised at primarily of carbon which take the
form of tubes, rods, cylinders, bundles, wafers, disks, sheets,
plates, planes, cones, slivers, granules, ellipsoids, wedges,
polymeric fibers, natural fibers, and other such objects which have
at least one characteristic dimension less than about 100 nm.
Preferred carbon nanostructures of the invention are nanotubes.
[0107] Nanotubes of the invention are generally about 1-200 nm in
diameter where the ratio of the length dimension to the narrow
dimension, i.e., the aspect ratio, is at least 5. In general, the
aspect ratio is between 10 and 2000. Carbon nanotubes are comprised
primarily of carbon atoms, however may be doped with other
elements, e.g., metals. The carbon-based nanotubes of the invention
can be either multi-walled nanotubes (MWNTs) or single-walled
nanotubes (SWNTs). A MWNT, for example, includes several concentric
nanotubes each having a different diameter. Thus, the smallest
diameter tube is encapsulated by a larger diameter tube, which in
turn, is encapsulated by another larger diameter nanotube. A SWNT,
on the other hand, includes only one nanotube.
[0108] Carbon nanotubes (CNT) may be produced by a variety of
methods, and are additionally commercially available. Methods of
CNT synthesis include laser vaporization of graphite (A. Thess et
al. Science 273, 483 (1996)), arc discharge (C. Journet et al.,
Nature 388, 756 (1997)) and HiPCo (high pressure carbon monoxide)
process (P. Nikolaev et al. Chem. Phys. Lett. 313, 91-97 (1999)).
Chemical vapor deposition (CVD) can also be used in producing
carbon nanotubes (J. Kong et al. Chem. Phys. Lett. 292, 567-574
(1998); J. Kong et al. Nature 395, 878-879 (1998); A. Cassell et
al. J. Phys. Chem. 103, 6484-6492 (1999); H. Dai et al. J. Phys.
Chem. 103, 11246-11255 (1999)).
[0109] Additionally CNT's may be grown via catalytic processes both
in solution and on solid substrates (Yan Li, et al., Chem. Mater.;
2001; 13(3); 1008-1014); (N. Franklin and H. Dai Adv. Mater. 12,
890 (2000); A. Cassell et al. J. Am. Chem. Soc. 121, 7975-7976
(1999)).
Peptide Generation
[0110] Peptides of the invention are generated randomly and then
selected against a population of carbon nanostructures for binding
affinity to CNT's. The generation of random libraries of 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, D. M., et al.,
Proc. Natl. Acad. Sci. USA 80(1): 31-35, 1983) yeast display (Chien
C T, et al., Proc Natl Acad Sci USA 1991 Nov. 1; 88(21): 9578-82)
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), 447468 (2001).
[0111] 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 protein on the
exterior of phage viron, while the DNA encoding the fusion residues
within the virion. This physical linkage between the displayed
protein and the DNA encoding it allows screening of vast numbers of
variants of proteins, 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 target. The eluted phage is then
amplified in vivo and the process repeated, resulting in 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.
[0112] Thus it is an object of the invention to provide a process
for generating a carbon nanostructure binding peptide comprising:
[0113] a) providing a library of phages expressing peptides in
solution; [0114] b) providing a population of carbon
nanostructures; [0115] c) contacting the phage of (a) with the
nanostructures of (b) for a time sufficient to permit binding of
the phage to the nanostructures and forming a phage-nanostructure
complex; [0116] d) removing unbound phage; [0117] e) contacting the
phage-nanostructure complex of (c) with a suitable bacterial host
whereby the bacteria are infected by the phage; [0118] f) growing
the infected bacteria of step (e) for a time sufficient to permit
replication of the phage and the expressed peptide; and [0119] g)
isolating the replicated phage and expressed peptide of step (f)
wherein the peptide binds carbon nanostructures. Peptide
Selection
[0120] After a suitable library of peptides has been generated they
are then contacted with an appropriate population of carbon
nanostructures or nanotubes. The nanotubes are presented to the
library of peptides typically while suspended in solution, although
it will be appreciated that CNT or peptides could also be
immobilized on a solid substrate to facilitate binding. In such an
embodiment suitable solid substrates will include but are not
limited to silicon wafers, synthetic polymer substrates, such as
polystyrene, polypropylene, polyglycidylmethacrylate, substituted
polystyrene (e.g., aminated or carboxylated polystyrene;
polyacrylamides; polyamides; polyvinylchlorides, etc.); glass,
agarose, nitrocellulose, and nylon.
[0121] A preferred solution is a buffered aqueous saline solution
containing a surfactant. A suitable solution is Tris-buffered
saline with 0.1% Tween 20. The solution can additionally be
agitated by any means in order to increase binding of the peptides
to the nanotubes.
[0122] Upon contact a number of the randomly generated peptides
will bind to the CNT's to form a peptide-nanotube complex. Unbound
peptide and CNT may be removed by washing (if immobilized) or by
any other means such as centrifugation, or filtering, etc. After
all unbound material is removed, peptides, having varying degrees
of binding affinities for CNT's may be fractionated by selected
washings in buffers having varying strengths of surfactants. The
higher the concentration of surfactant in the wash buffer, the
higher the stringency of selection. Increasing the stringency used
will increase the required strength of the bond between the peptide
and nanotube in the peptide-nanotube complex.
[0123] A number of materials may be used to vary the stringency of
the buffer solution in peptide selection including but not limited
to acidic pH 1.5-3; basic pH 10-12.5; high salt concentrations such
as MgCl2 3-5 M, LiCl 5-10 M; water; ethylene glycol 25-50%; dioxane
5-20%; thiocyanate 1-5 M; guanidine 2-5 M; urea 2-8 M; various
concentrations of different surfactants such as SDS (sodium dodecyl
sulfate), DOC (sodium deoxycholate), Nonidet P-40, Triton X-100,
Tween 20.RTM. wherein Tween 20.RTM. is preferred. The materials can
be prepared in buffer solutions including but not limited to
Tris-HCl, Tris-borate, Tris-acidic acid, triethylamine, phosphate
buffer, glycine-HCl wherein 0.25M glycine-HCl solution is
preferred.
[0124] It will be appreciated that peptides having greater and
greater binding affinities for the CNT substrate may be eluted by
repeating the selection process using buffers with increasing
stringencies.
[0125] The eluted peptides can be identified, sequenced, and
produced by any means known in the art.
Carbon Nanotube Binding Peptides
[0126] Peptides of the invention selected by the above process have
been identified. A large number of peptides having particularly
high binding affinities to carbon nanotubes were isolated having
the amino acid sequences as set forth in SEQ ID NOs:1-24 and
35-177
[0127] It will be appreciated by the skilled artisan that the
invention is not limited to these specific sequences but will
include amino acid sequences comprising chemically equivalent amino
acid substitutions that do not interfere with the ability of the
peptide to bind CNT's. So for example, the chemically equivalent
substitutions for each of the amino acids in SEQ ID NO:14 are
detailed in the following table: TABLE-US-00002 SEQ ID NO: 14 His
Trp Ser Ala Trp Trp Ile Arg Ser Asn Gln Ser Equivalent Lys Phe Pro
Ser Phe Phe Lys Pro Asp Asp Pro Amino Acids Arg Tyr Ala Pro Tyr Tyr
His Ala Glu Asn Ala Thr Thr Thr Gln Glu Thr Gly Gly Gly Gly
[0128] Alignment and analysis of the selected peptides of the
invention suggests that the carbon nanostructure or nanotube
binding properties are related to the secondary characteristics of
the peptide. For example a simple pendant model was developed for
the peptides of the instant invention, which accounts for
hydrophilicity or hydrophobicity. It demonstrates that all of the
consensus sequences are essentially symmetric
surfactants--hydrophilic on the ends and hydrophobic in the middle.
The model describes the degree of hydrophilicity or hydrophobicity
of an amino acid pendant group by classifying all pendant groups as
either hydrophilic (h=-1) or hydrophobic (h=1). Side chains which
are either basic, acidic or uncharged polar are be hydrophilic
while side chains that are nonpolar are hydrophobic. Several of the
peptides selected by the methods of the invention are modeled
below: TABLE-US-00003 (SEQ ID NO:1) H A H S Q W W H L P Y R -1 1 -1
-1 -1 1 1 -1 1 1 -1 -1 (SEQ ID NO:13) H W K H P W G A W D T L -1 1
-1 -1 1 1 1 1 1 -1 -1 1 (SEQ ID NO:14) H W S A W W I R S N Q S -1 1
-1 1 1 1 1 -1 -1 -1 -1 -1 (SEQ ID NO:8) H N W Y H W W M P H N T -1
-1 1 -1 -1 1 1 1 1 -1 -1 -1
These peptides were selected over a broad range of detergent
concentrations (0.6%-3%) and yet show the same pattern of
hydrophilicity and hydrophobicity. With a few exceptions, the h=-1
are predominantly on the ends and h=1 are concentrated in the
middle.
[0129] It is thus an object of the invention to provide a carbon
nanotube binding peptides having the general structure: N-M-C
Wherein:
[0130] N is the N-terminal portion of the peptide having about 4
amino acids, 75% of which are hydrophilic;
[0131] M is the median portion of the peptide having about 4 amino
acids, 75% of which are hydrophobic; and
[0132] C is the C-terminal portion of the peptide having about 4
amino acids 75% of which are hydrophilic.
Peptide Production by Recombinant Methods
[0133] Once a peptide having suitable binding properties is
identified it may be produced recombinantly in large quantities.
Genes encoding nanotube binding peptides may be produced in
heterologous host cells, particularly in the cells of microbial
hosts.
[0134] Preferred heterologous host cells for expression of nanotube
binding peptides 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 of
transcription, translation and the protein biosynthetic apparatus
is 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.
[0135] 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 baculaviruses, 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 a regulatory elements may also
be present in the vector, for example, enhancer sequences. Any of
these could be used to construct chimeric genes for production of
the any of the nanotube binding peptides. These chimeric genes
could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the
peptides.
[0136] 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 tetracyclin or ampicillin
resistance in E. coli.
[0137] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator or control element, so that Initiation control regions
or promoters, which are useful to drive expression of the instant
ORF's in the desired host cell are numerous and familiar to those
skilled in the art. Virtually any promoter capable of driving these
genes is suitable for 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.
[0138] 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.
[0139] The vector containing the appropriate DNA sequence as here
in above described, 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.
[0140] 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 host production host. Methods for choosing appropriate signal
sequences are well known in the art (see for example EP 546049; 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.
Nano-Device Fabrication
[0141] The carbon nanotube binding peptides (CNBP) of the instant
invention could be one element in an entity with bi-, tri- (or
higher) binding functionality. A CNBP can be depicted graphically
as shown below, ##STR1## where the "C" is suggestive of a carbon
nanotube and a binding functionality depicted by ".OR right.".
However, despite its being drawn at one end, it should be
interpreted as a collective, not localized, property of the peptide
sequence. The overall entity would be constructed by "fusion" of
the CNBP with another body, depicted by "B", with a binding
functionality depicted by ">", and the combination is
represented graphically below ##STR2##
[0142] This is meant to represent a minimal example; the fusion
could create higher order (enumerative) functionality. Examples of
B include but are not limited to a DNA binding protein, a metallic
electrode, for example Au bound directly to an amino-acid residue
like cysteine, or a hard (e.g., Si or SiO.sub.2) substrate for
immobilization of the CNBP.
[0143] Directed self-assembly of carbon nanotubes into useful
structures could be achieved by combining the binding of CNBP with
a pre-patterned substrate. For example, if the binding
functionality "B" was a series of cysteine residues, the sequence
of (a) preparation of a dilute suspension of carbon nanotubes, (b)
functionalization of selected types by CNBP, and (c) washing over a
substrate with patterned Au electrodes would result in the
attachment of carbon nanotubes to metal electrodes via the peptide,
within distances of relevance to nano-electronic devices. Because
of the diversity of the bio-chemical toolkit in combining elements
to obtain higher order functionality, many other such methods can
be conceived, once the fundamental binding motifs have been
identified.
[0144] A major obstacle to the use of carbon nanotubes in a variety
of applications is the fact that all manufacturing processes
produce a mixture of entangled tubes. Individual tubes in the
product differ in diameter, chirality, and number of walls.
Moreover, long tubes show a strong tendency to aggregate into
"ropes". These ropes are formed due to the large surface areas of
nanotubes and can contain tens to hundreds of nanotubes in one
rope. Furthermore, the structure of individual tubes varies widely
from armchair, zig-zag or other chiral forms which coexist in the
material and their electrical properties also vary dramatically
accordingly (metallic or semi-conductive). Therefore, a need exists
for the isolation of a single form (such as armchair, zig-zag or a
chiral form) of carbon nanotubes.
[0145] Existing methods for separating such product, for example
acid washing, ultra-sonification, and use of surfactants, is
non-specific with respect to the type of nanotube. Because peptide
binding is usually highly specific, a major utility of a CNBP is to
effect specific separation. One possible method would use dilute
suspensions of carbon nanotubes separated by having them flow over
substrates patterned with different types of binding CNBP's. One
would choose to order the patterning based on strength and
specificity of binding, i.e., strongly selective binding peptides
would be positioned to act on the mixture in advance of less
specific ones. Many other ways to achieve separation can be
conceived. If "B" binds to a magnetic particle, the joint entity
could be used in one stage of a continuous flow to bind to carbon
nanotubes, while in another stage the bound nanotubes could be
separated magnetically.
[0146] Thus it is an object of the invention to provide a method of
dispersing a population of carbon nanotube ropes comprising: [0147]
a) providing a population of carbon nanotubes in solution in rope
formation; and [0148] b) contacting the population of carbon
nanotubes of step (a) with a carbon nanotube binding peptide having
the general structure: N-M-C [0149] wherein:
[0150] N is the N-terminal portion of the peptide having about 4
amino acids, 75% of which are hydrophilic;
[0151] M is the median portion of the peptide having about 4 amino
acids, 75% of which are hydrophobic; and
[0152] C is the C-terminal portion of the peptide having about 4
amino acids 75% of which are hydrophilic;
[0153] whereby the carbon nanotube ropes are dispersed.
[0154] One of skill in the art will appreciate that it will be
useful to sort populations of nanotubes to select for various
binding properties. It is contemplated that CNT-binding peptides or
isolated phage expressing a CNT binding peptide, may be used for
this purpose. For example, CNT-binding peptides or phages
expressing the same, having an affinity to a specific population of
CNT's may be immobilized on a solid substrate and then contacted
with a mixed population of CNT's. The desired CNT's will bind to
the immobilized peptides or phage and the undesired CNT's may be
washed free. Alternatively, solid substrates such as beads or
microspheres may be coated with CNT-binding peptides or phage and
used to assemble CNT's. Materials suitable as solid supports may be
made of synthetic polymers such as polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), nylon, poly(vinyl butyrate), or other materials
such as glass, ceramics, metals, and the like. These materials be
used as films, microtiter plates, wells, beads, slides, particles,
pins, pegs, or membranes.
[0155] Accordingly the invention a method for assembling carbon
nanotubes comprising contacting a solid substrate coated with at
least one species of carbon nanotube binding peptide with a
population of carbon nanotubes whereby the carbon nanotubes bind to
the coated substrate and are assembled.
[0156] It will be appreciated by the skilled artisan that
patterning of CNT-binding peptides on a particular solid support
will be a useful technique in the design and fabrication of
nanodevices. In some instances patterning may be achieved by
partially and selectively masking portions of the support with
materials that repel or have no affinity for CNT's. A variety of
materials may be used for this purpose, however non-CNT binding
peptides of similar physical characteristics to CNT-binding
peptides will be particularly suitable. Non-CNT binding peptides
are easily selected and identified in the early rounds of any
selection process for CNT-binding peptides. These may be used to
mask a solid support to effect the pattering of CNT binding on the
support. Several examples provided herein include the peptides set
forth in SEQ ID NO:28 and 34.
EXAMPLES
[0157] 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 usages and conditions.
General Methods
[0158] 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, (1989) (Maniatis) and 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, pub. by Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0159] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
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.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.)
unless otherwise specified.
Preparation of Phage Library
[0160] The phage library used in the present invention was
purchased from New England BioLabs (catalog number E8110S, Ph.D.-12
Phage Display Peptide Library Kit). The kit is based on a
combinatorial library of random peptide 12-mers fused to a minor
coat protein (pIII) of M13 phage. The displayed peptide 12mer is
expressed at the N-terminus of pill, i.e. after the signal peptide
is cleaved the first residue of the coat protein is the fist
residue of the displayed peptide. The library contains
2.7.times.10.sup.9 (100 .mu.l) variants in the displayed epitope. A
volume of 10 .mu.l contains about 55 copies of each peptide
sequence. To avoid introduce bias into the library, each initial
round of experiments were carried out using the original library
provided by the manufacture.
Sequencing of Phases
[0161] Random M13 phage plaques were picked and single plaque
lysates were prepared following manufacture's instruction (New
England Labs, Beverly, Mass.). The single stranded phage genome DNA
was purified with Qiagene kit (QIAprep Spin M13 kit, Cat. No.
27704). The single stranded DNA were sequenced with -96 gill
sequencing primer (5'-CCCTCATAGTTAGCGTAACG-3'). SEQ ID NO:33 The
displayed peptide is located immediately after the signal peptide
of gene III.
Multi-Sequence Analysis
[0162] Sequences from the phage display experiment were analyzed
using software DNA Star (version 5.02) or ClustalW according to
software instructions. The alignment provides similarities among
the selected sequences and predict important functions of certain
amino acid residues.
Example 1
Preparation of Carbon Nanotubes
[0163] Carbon nanotubes designated CNT-7 were obtained from
Yet-Ming Chiang, Department of Materials Science, MIT, Cambridge,
Mass. The nanotubes were prepared by heating SiC (silicon carbide)
at 1700.degree. C. under vacuum. Silicon "evaporated" from the
sample and left behind the carbon, which formed folded structures
of carbon including nanotubes.
[0164] Single-walled carbon nanotubes were purchased from CNI
(Carbon Nanotechnology Incorporated, Houston, Tex.). These
nanotubes were produced by a laser oven technique or a HiPCO
(high-pressure carbon monoxide) process (P. Nikolaev et al. Chem.
Phys. Lett. 313, 91-97 (1999)).
[0165] The CNT-7 sample contained various carbon structures
including multi-wall and single-wall carbon nanotubes whereas CNI
samples were mostly single wall carbon nanotubes.
Example 2
Selection of Carbon Nanotube Binding Peptides
[0166] CNT-7 and CNI carbon nanotubes were suspended in
Tris-Buffered-Saline with 0.1% Tween 20 (TBS-T) at a concentration
of 1 mg/ml. The carbon nanotube solution was then sonicated by a
Branson Sonifier model 450 (Branson Sonic Power Co., Danbury,
Conn.) with power output setting between 4 and 5, duty cycle 70-80%
for three times. Ten microliters of M13 phage library (containing
about 10.sup.11 phage) were added to 1 ml of carbon nanotubes. The
mixture was incubated at room temperature with mild agitation for
60 minutes. Unbound phages were separated from the nanotube sample
by high speed spin at 14,000 rpm (16,110.times.g) in an Eppendorf
5415C centrifuge (Brinkmann Instruments Inc., Westbury, N.Y.) for
10 minutes. Subsequently the phage/nanotube complex was washed 10
times each with 1 ml TBS-T in which concentration of Tween-20
increases according to the cycles of selection, as shown in the
data below. For example, in one experiment the Tween 20
concentration was increased from 0.2% in round one, to 0.3% in
round two, to 0.4% in round 4, to 0.5% in round 5, to 0.6% in round
6, to 0.7% in round 7, to 1% in round 8, to 2% in round 9, 3% in
round 10, 6% in round 11 and 10% in round 12. After the last
(tenth) washing step, the bound phages were eluted off by
incubating with 0.5 ml of 0.25 M glycine-HCl, pH 3.0 for 10-15
minutes at room temperature. The phages and nanotubes were
separated by centrifuging at 14,000 rpm (16,110.times.g) for 10
minutes, with the cleared supernatant containing the eluted phages.
The presence and concentration of phages in the supernatant were
determined by phage titering. Once the sample confirmed the
presence of phages, they were used to inoculate E. coli for phage
amplification, and the amplified phage sample was used as the
"pool" for next round. In a typical experiment, the entire eluent
was added to a 20 ml E. coli culture at early log phase. The
culture was further incubated for 4.5 hours at 37.degree. C. to
allow phage to propagate. At the end of the incubation, the
cultures were spin at 16,000.times.g for 10 minutes at 4.degree. C.
The phages in the cleared supernatant were precipitated with
PEG/NaCl at 4.degree. C. After centrifugation, the phages were
resuspended in 200 .mu.l PBS and the concentration was determined
by titering. This sample is used subsequently as the stock for the
next round experiment. To carry out the next round experiment,
10.sup.11 phages were used as input "pool" and the selection
process was repeated as described above with increased stringency
for washing, i.e. increased concentration of Tween-20. Useful
peptides were obtained by selection at detergent concentrations of
0.5% and higher and the amino acid sequences of these peptides are
shown in Table 1. TABLE-US-00004 TABLE 1 M13 peptide sequences for
CNT-7 SEQ ID NO: Sequence 1. DPHHHWYHMHQH 2. HAHSQWWHLPYR 3.
HAHSRRGHIQHR 4. HCHHPWGAWHTL 5. HCWNQWCSRHQT 6. HGNWSYWWSKPS 7.
HHWHHWCMPHKT 8. HNWYHWWMPHNT 9. HNWYRWCIRHNN 10. HRWYRWSSRNQT 11.
HSSWWLALAKPT 12. HWCAWWISSNQS 13. HWKHPWGAWDTL 14. HWSAWWIRSNQS 15.
HWSPWHRPWYQP 16. HYSWYSTWWPPV 17. HYWWRWWMPNQT 18. KCHSRHDHIHHH 19.
KSLSRHDHIHHH 20. KSRSRHDEIHHH 21. KYRSRHDHIHHH 22. QWHSRHDHIHHH 35.
HNWYHWWPHNT 36. HWYKPYHFQSLT 37. SVSVGMKPSPRP 38. EAHPQTLGWQRP 39.
HNAYWHWPPSMT
[0167] Binding of these peptides to CNT's is further confirmed by
TEM micrographs as shown in FIG. 1, illustrating a number of the
subject peptides bound to a single walled CNT.
Example 3
Structural and Functional Characterization of Nanotube-Binding
Peptides
[0168] The following example illustrates the importance of
conserved amino acids to the binding affinity of peptides for
carbon nanotubes.
[0169] Alignment of the selected peptide sequences suggests
strongly that histidine at position 1 and tryptophan at position 6
are important for binding. Further analysis of more than one
hundred phage clones, shown below in Table 3, revealed that His and
Trp are two dominant amino acids in the composition of peptides
selected by the display. TABLE-US-00005 TABLE 3 Change Number % by
% by Original relative to Amino Acids count weight frequency
library (%) original (%) Charged: RKHYCDE 391 36.03 33.59 (SEQ ID
NO: 29) Acidic: DE 37 2.84 3.18 Basic: KR 89 8.51 7.65 Polar:
NCQSTY 305 22.51 26.20 (SEQ ID NO: 30) Hydrophobic: AILFW 376 37.62
32.3 (SEQ ID NO: 31) A 57 2.69 4.90 6.0 -- C 6 0.41 0.52 0.5 0.52 D
35 2.67 3.01 2.8 3.01 E 2 0.17 0.17 3.1 0.17 F 5 0.49 0.43 3.3 0.43
G 30 1.13 2.58 2.6 2.58 H 217 19.73 18.64 6.3 -- I 27 2.03 2.32 3.4
2.32 K 38 3.23 3.26 2.8 3.26 L 55 4.13 4.73 9.3 4.73 M 25 2.17 2.15
2.6 2.15 N 66 4.99 5.67 4.6 5.67 P 85 5.47 7.30 12.2 7.30 Q 37 3.14
3.18 5.1 3.18 R 51 5.28 4.38 4.7 4.38 S 97 5.60 8.33 10.0 8.33 T 57
3.82 4.90 11.1 4.9 V 6 0.39 0.52 3.9 0.52 W 226 27.90 19.42 2.2 --
Y 42 4.54 3.61 3.6 3.61 B 0 0 0 Z 0 0 0 X 0 0 0 . Ter 0 0 0 1. Data
is from analysis of 100 clones 2. Original library data is adapted
from manufacturer's manual and is from analysis of 104 clones
[0170] Site-directed mutagenesis was used to introduce mutations in
peptides SEQ ID NO:13 (HWKHPWGAWDTL) and SEQ ID NO:14
(HWSAWWIRSNQS) Trp->Ser at position 6, to produce peptides
HWKHPSGAWDTL (SEQ ID NO:26) and HWSAWSIRSNQS (SEQ ID NO:27),
respectively. The phages carrying these mutations were assayed for
their binding activity against CNT-7 at detergent concentration
0.4% as described above. The mutation at Trp6 reduced binding to
CNT-7 for both peptides. The data is shown in Table 4 below. The
number of plaque forming units is charted for a control peptide
(LPPSNASVADYS) SEQ ID NO:28 and peptides SEQ ID NO:13, 14 and
mutant peptides SEQ ID NO: 26 and 27. The binding data shown in
Table 4 confirms the critical role of Trp in binding to nanotubes.
TABLE-US-00006 TABLE 4 Phage Pfu SEQ ID NO: 26 4.58 .times.
10.sup.6 SEQ ID NO: 27 5.4 .times. 10.sup.6 SEQ ID NO: 28 6.7
.times. 10.sup.6 SEQ ID NO: 13 15.9 .times. 10.sup.6 SEQ ID NO: 14
59.2 .times. 10.sup.6
Example 4
Effect of Peptide Binding on Populations of Nanotubes
[0171] The following example illustrates the ability of carbon
nanotube binding peptides to disentangle carbon nanotube
"ropes".
[0172] Experiments were carried out with synthetic peptides and
single-wall carbon nanotubes (CNI/Laser oven) and binding peptides
sequence HWKHPWGAWDTLGGG [SEQ ID NO: 25]. This peptide was selected
as described in Example 2 and represents the peptide as set forth
in SEQ ID NO:13, with the addition of a poly-glycine tail.
[0173] At a concentration of 4 mg/ml peptide of SEQ ID NO:25 was
seen to disperse the nanotube ropes as examined by HRTEM whereas
the mutant peptide SEQ ID NO:26, containing a polyglycine tail
HWKHPSGAWDTLGGG [SEQ ID NO:32] and a control peptide SEQ ID NO:28
did not disperse the nanotube at the same concentration. The
results are shown in FIG. 2. Panel A of FIG. 2 is an electron
micrograph of nanotubes ropes untreated with any peptide. Panel B
of FIG. 2 is an electron micrograph of single walled nanotubes
after treatment with the carbon nanotube binding peptide of SEQ ID
NO:13 showing dispersement of the nanotubes. Panel C of FIG. 2 is
an electron micrograph of single walled nanotubes after treatment
with the peptide of SEQ ID NO:28, a control peptide having little
or no nanotube binding affinity.
Example 5
Graphite-Cleaned Binding Peptides
[0174] In order to find peptides with specific binding to carbon
nanotubes, phage display experiments were performed as described in
Example 2 on CNT-7 carbon nanotube substrates using a
"graphite-cleaned" phage library. The graphite-cleaned phage
library was generated by first washing the complete phage library
on a pyrolytic graphite substrate. The washed or cleaned library
was thus denuded of phage that would bind to Graphite. Highly
ordered pyrolytic Graphite (HOPG SPI-2, SPI Supplies, West Chester,
Pa.) was attached to a petri dish and a fresh layer of graphite was
exposed using a Scotch tape. About 10.sup.11 pfu M13 phage in
TBS-0.1% Tween-20.RTM. was added to the graphite substrate and
allowed to sit for binding for 45-60 minutes at room temperature.
Unbound phages were washed away with excess amount of (TBS-T) at
defined concentrations of Tween-20.RTM.. Bound phages were eluted
with Glycine-HCl buffer at pH 2.3. The unbound phage
(graphite-cleaned library) were then used to perform phage display
experiments on CNT-7 as described in Example 2. Individual phages
were isolated and DNA sequences were obtained using standard
molecular biology methods described above.
[0175] After four rounds of phage display on CNT-7 with the
graphite-cleaned library (round 4 with 0.5% concentration of
Tween-20.RTM.), two consensus sequences emerged. These are:
[0176] HHHHLRHPFWTH (SEQ ID NO:23) and WPHHPHMHTIR (SEQ ID
NO:24)
[0177] The implication is that the binding of these sequences is
specific to the CNT-7, as compared to a graphitic clone. The
significance of the finding is in the close relationship between
the graphene sheet that bounds freshly cleaved graphite, and the
surface of carbon nanotubes. Carbon nanotube surfaces are
essentially curved or graphene sheets. As such, objects may bind
both to carbon nanotubes and to graphite. Additional significance
may be attached to those whose binding discriminates between the
two. This result illustrates that peptides can recognize different
allotropes of carbon.
Example 6
Peptide Facilitated Binding of CNT to Microspheres
[0178] Example 6 illustrates that microspheres coated with CNT
binding peptides are effective in binding single walled nanotubes
and forcing assembly of the microspheres.
[0179] Preparation of Phage-Coated Microspheres.
[0180] Purified phage clones were amplified. Anti-mouse antibody
IgG-coated microspheres (seven microns in diameter from Bangs
Laboratories, Inc, 9025 Technology Drive, Fishers Ind. 46038-2886)
were coated with an anti-M13 monoclonal antibody (Amersham
pharmacia biotech Inc. 800 Centennial Avenue PO Box 1327 Piscataway
N.J. 08855). Purified phage clones were coated onto these
microspheres in TBS buffer. The phage-coated microspheres were
incubated overnight with 10% Triton-X-165 dispersed SWNTs 7.5
.mu.g/ml in a dialysis tube against 1 L of TBS buffer containing 10
grams of Amberlite XAD-4 (Sigma, P.O. Box 14508 ST. Louis, Mo.
63178). The microspheres were then washed three times with water.
The beads were examined under SEM.
[0181] Preparation of Peptide-Coated Microspheres.
[0182] Selected sequences were synthesized as free peptides,
including, NH.sub.2-HWKHPWGAWDTLGGG-COOH (SEQ ID NO:25) and
NH.sub.2-HWKHPWGAWDTL-COOH (SEQ ID NO:13). Amino-modified
microspheres (0.66 microns in diameter from Bangs Laboratories,
Inc, 9025 Technology Drive, Fishers Ind. 46038-2886) were
cross-linked to synthetic peptides at the C terminus through an EDC
linker (Pierce Inc. 3747 N. Meridian Road, P.O. Box 117, Rockford,
Ill. 61105 Sigma, P.O. Box 14508 ST. Louis, Mo. 63178). The
peptide-coated microspheres were incubated overnight with 10%
Triton-X-165 dispersed SWNT 7.5 .mu.g/ml in a dialysis tube against
1 L of TBS buffer containing 10 grams of Amberlite XAD-4. The
microspheres were then washed three times with TBS buffer. The
beads were examined under SEM.
[0183] Results of contacting CNT's with microspheres either coated
with CNT binding phage or isolated peptide are illustrated in FIG.
3a-d. FIG. 3(a) shows the surface of a microsphere coated with a
control phage clone expressing peptide sequence
NH.sub.2-IDVESYKGTSMP-COOH. (SEQ ID NO:34). Clearly, there is no
association of the carbon nanotubes with this surface. FIG. 3(b)
shows the surface of a microsphere coated with the binding phage
clone sequence NH.sub.2--HWKHPWGAWDTL-COOH (SEQ ID NO:13). It
demonstrates strong association between the phage and nanotube
bundles. Similar results have been obtained with other
nanotube-binding phage clones. FIG. 3(c), coated with the control
peptide NH.sub.2-LPPSNASVADYSGGG-COOH (SEQ ID NO:28), shows no
association of microspheres with nanotubes. Indeed, the suspension
of microspheres remained highly dispersed. FIG. 3(d) shows strong
association between the microspheres coated with the binding
peptide NH.sub.2--HWKHPWGAWDTL-COOH (SEQ ID NO:13) and nanotubes.
Essentially, the nanotubes cross-linked the microspheres, resulting
in a loss of dispersion of the microspheres and formation of large
clusters of microspheres.
Example 7
CMBP Generated to a Variety of Carbon Nanotube Substrates
[0184] This example illustrates that carbon nanotube binding
peptides may be generated to a variety of carbon nanotube
substrates including those made by the HiPCo process and those that
have undergone various cleaning processes.
[0185] A series of experiments to select carbon nanotube binding
peptides were performed as described in Example 2. The first
substrate used was SWNTs from CNI prepared using the HiPCo process
that was prepared only by acid cleaning, dispersion in toluene and
drying to form a mat. Peptides resulting after the selection
process are listed in SEQ. ID Nos:39-85.
[0186] The second substrate used in the selection process as
described in Example 2 were MWNTs obtained from Yet-Ming Chiang,
Department of Materials Science, MIT, Cambridge, Mass. Peptides
that were selected are listed in SEQ. ID Nos:1-4, 6, 8, 10-13, 15,
16, 18-22, 28, 36, 92, 114-147, and 177. The above example was
repeated used fresh MWNTs obtained from the same source. Resulting
peptides are listed in SEQ. ID Nos:94-113 and 177.
[0187] Graphite-cleaned MWNTs obtained from Yet-Ming Chiang,
Department of Materials Science, MIT, Cambridge, Mass. prepared as
described in Example 5 and were also used as substrates. Resulting
peptides are listed in SEQ. ID Nos:148-176.
Sequence CWU 1
1
177 1 12 PRT artificial sequence generated by phage display 1 Asp
Pro His His His Trp Tyr His Met His Gln His 1 5 10 2 12 PRT
artificial sequence generated by phage display 2 His Ala His Ser
Gln Trp Trp His Leu Pro Tyr Arg 1 5 10 3 12 PRT artificial sequence
generated by phage display 3 His Ala His Ser Arg Arg Gly His Ile
Gln His Arg 1 5 10 4 12 PRT artificial sequence generated by phage
display 4 His Cys His His Pro Trp Gly Ala Trp His Thr Leu 1 5 10 5
12 PRT artificial sequence generated by phage display 5 His Cys Trp
Asn Gln Trp Cys Ser Arg His Gln Thr 1 5 10 6 12 PRT artificial
sequence generated by phage display 6 His Gly Asn Trp Ser Tyr Trp
Trp Ser Lys Pro Ser 1 5 10 7 12 PRT artificial sequence generated
by phage display 7 His His Trp His His Trp Cys Met Pro His Lys Thr
1 5 10 8 12 PRT artificial sequence generated by phage display 8
His Asn Trp Tyr His Trp Trp Met Pro His Asn Thr 1 5 10 9 12 PRT
artificial sequence generated by phage display 9 His Asn Trp Tyr
Arg Trp Cys Ile Arg His Asn Asn 1 5 10 10 12 PRT artificial
sequence generated by phage display 10 His Arg Trp Tyr Arg Trp Ser
Ser Arg Asn Gln Thr 1 5 10 11 12 PRT artificial sequence generated
by phage display 11 His Ser Ser Trp Trp Leu Ala Leu Ala Lys Pro Thr
1 5 10 12 12 PRT artificial sequence generated by phage display 12
His Trp Cys Ala Trp Trp Ile Ser Ser Asn Gln Ser 1 5 10 13 12 PRT
artificial sequence generated by phage display 13 His Trp Lys His
Pro Trp Gly Ala Trp Asp Thr Leu 1 5 10 14 12 PRT artificial
sequence generated by phage display 14 His Trp Ser Ala Trp Trp Ile
Arg Ser Asn Gln Ser 1 5 10 15 12 PRT artificial sequence generated
by phage display 15 His Trp Ser Pro Trp His Arg Pro Trp Tyr Gln Pro
1 5 10 16 12 PRT artificial sequence generated by phage display 16
His Tyr Ser Trp Tyr Ser Thr Trp Trp Pro Pro Val 1 5 10 17 12 PRT
artificial sequence generated by phage display 17 His Tyr Trp Trp
Arg Trp Trp Met Pro Asn Gln Thr 1 5 10 18 12 PRT artificial
sequence generated by phage display 18 Lys Cys His Ser Arg His Asp
His Ile His His His 1 5 10 19 12 PRT artificial sequence generated
by phage display 19 Lys Ser Leu Ser Arg His Asp His Ile His His His
1 5 10 20 12 PRT artificial sequence generated by phage display 20
Lys Ser Arg Ser Arg His Asp Glu Ile His His His 1 5 10 21 12 PRT
artificial sequence generated by phage display 21 Lys Tyr Arg Ser
Arg His Asp His Ile His His His 1 5 10 22 12 PRT artificial
sequence generated by phage display 22 Gln Trp His Ser Arg His Asp
His Ile His His His 1 5 10 23 12 PRT artificial sequence generated
by phage display 23 His His His His Leu Arg His Pro Phe Trp Thr His
1 5 10 24 12 PRT artificial sequence generated by phage display 24
Trp Pro His His Pro His Ala Ala His Thr Ile Arg 1 5 10 25 15 PRT
artificial sequence generated by phage display 25 His Trp Lys His
Pro Trp Gly Ala Trp Asp Thr Leu Gly Gly Gly 1 5 10 15 26 12 PRT
artificial sequence generated by phage display 26 His Trp Lys His
Pro Ser Gly Ala Trp Asp Thr Leu 1 5 10 27 12 PRT artificial
sequence generated by phage display 27 His Trp Ser Ala Trp Ser Ile
Arg Ser Asn Gln Ser 1 5 10 28 12 PRT artificial sequence generated
by phage display 28 Leu Pro Pro Ser Asn Ala Ser Val Ala Asp Tyr Ser
1 5 10 29 7 PRT artificial sequence generated by phage display 29
Arg Lys His Tyr Cys Asp Glu 1 5 30 6 PRT artificial sequence
generated by phage display 30 Asn Cys Gln Ser Thr Tyr 1 5 31 5 PRT
artificial sequence generated by phage display 31 Ala Ile Leu Phe
Trp 1 5 32 15 PRT artificial sequence generated by phage display 32
His Trp Lys His Pro Ser Gly Ala Trp Asp Thr Leu Gly Gly Gly 1 5 10
15 33 20 PRT artificial sequence generated by phage display 33 Cys
Cys Cys Thr Cys Ala Thr Ala Gly Thr Thr Ala Gly Cys Gly Thr 1 5 10
15 Ala Ala Cys Gly 20 34 12 PRT artificial sequence generated by
phage display 34 Ile Asp Val Glu Ser Tyr Lys Gly Thr Ser Met Pro 1
5 10 35 11 PRT artificial sequence generated by phage display 35
His Asn Trp Tyr His Trp Trp Pro His Asn Thr 1 5 10 36 12 PRT
artificial sequence generated by phage display 36 His Trp Tyr Lys
Pro Tyr His Phe Gln Ser Leu Thr 1 5 10 37 12 PRT artificial
sequence generated by phage display 37 Ser Val Ser Val Gly Met Lys
Pro Ser Pro Arg Pro 1 5 10 38 12 PRT artificial sequence generated
by phage display 38 Glu Ala His Pro Gln Thr Leu Gly Trp Gln Arg Pro
1 5 10 39 12 PRT artificial sequence generated by phage display 39
His Asn Ala Tyr Trp His Trp Pro Pro Ser Met Thr 1 5 10 40 12 PRT
Artificial sequence generated by phage display 40 Ala Glu Pro Trp
Ala Ser Val Ser Thr Pro Pro Pro 1 5 10 41 12 PRT Artificial
sequence generated by phage display 41 Ala His Arg Ser Asp Phe Trp
Arg Pro Phe Pro Thr 1 5 10 42 12 PRT Artificial sequence generated
by phage display 42 Ala Leu Pro Arg Asn Asp Leu Ser Asp Ala Ala Ser
1 5 10 43 12 PRT Artificial sequence generated by phage display 43
Ala Thr Ser Thr Phe Trp Pro Arg Ala Phe Pro Ala 1 5 10 44 12 PRT
Artificial sequence generated by phage display 44 Asp Arg Val Pro
Ile Gln Pro Trp Thr Ala Pro Arg 1 5 10 45 12 PRT Artificial
sequence generated by phage display 45 Phe Gly Asn Ser Asp Lys Leu
Gln Thr Arg Ala Phe 1 5 10 46 12 PRT Artificial sequence generated
by phage display 46 Phe His Lys Ala Pro Lys Ser Pro Gly Met Pro Thr
1 5 10 47 12 PRT Artificial sequence generated by phage display 47
Phe His Arg His Gln Glu Met Thr Ala Thr Val His 1 5 10 48 12 PRT
Artificial sequence generated by phage display 48 Phe Pro Leu Arg
Pro Val Glu Val Lys Asp Ala Ser 1 5 10 49 12 PRT Artificial
sequence generated by phage display 49 Gly Leu Pro Glu Met Arg Leu
Pro Leu Val Pro Pro 1 5 10 50 12 PRT Artificial sequence generated
by phage display 50 Gly Gln Thr Ile Pro Val Asp Lys Thr Gln Ser Pro
1 5 10 51 12 PRT Artificial sequence generated by phage display 51
His Ala His Ser Trp Pro Pro Ala His Gln Leu His 1 5 10 52 12 PRT
Artificial sequence generated by phage display 52 His Phe Pro Leu
Ser Ser Asn Lys Val Pro Arg Ala 1 5 10 53 12 PRT Artificial
sequence generated by phage display 53 His Phe Ser Ser Thr Leu Ser
Leu Gln Glu Leu Asp 1 5 10 54 12 PRT Artificial sequence generated
by phage display 54 His Ile Lys Ile Gln Pro Arg Ala Pro Val Phe Met
1 5 10 55 12 PRT Artificial sequence generated by phage display 55
His Lys Pro His Leu Tyr Asn Lys Pro Thr Phe Thr 1 5 10 56 12 PRT
Artificial sequence generated by phage display 56 His Leu Lys Met
Pro Lys Phe Ala His Pro Asn Leu 1 5 10 57 12 PRT Artificial
sequence generated by phage display 57 His Leu Pro Met Thr Tyr Ser
Ala Thr Asn Pro Val 1 5 10 58 12 PRT Artificial sequence generated
by phage display 58 His Asn Lys Pro His His Phe Pro Arg Leu Leu Thr
1 5 10 59 12 PRT Artificial sequence generated by phage display 59
His Pro Met Val Glu Asn Thr Val Ser Ser Trp Thr 1 5 10 60 12 PRT
Artificial sequence generated by phage display 60 His Pro Thr Gln
Lys Asn Val His Pro Phe Arg Ser 1 5 10 61 12 PRT Artificial
sequence generated by phage display 61 His Ser Ser Pro His Phe Ser
Arg His Gly Leu Leu 1 5 10 62 12 PRT Artificial sequence generated
by phage display 62 His Thr Ile Pro Thr Ile Ser Thr His Phe Trp Asp
1 5 10 63 12 PRT Artificial sequence generated by phage display 63
His Thr Lys Gln Ile Pro Arg His Ile Tyr Ser Ala 1 5 10 64 12 PRT
Artificial sequence generated by phage display 64 Lys Thr Leu Tyr
Leu Pro Asn Ser Leu Arg Leu His 1 5 10 65 12 PRT Artificial
sequence generated by phage display 65 Lys Tyr Gly Asp Pro Leu Ser
Leu Thr Trp Gly Arg 1 5 10 66 12 PRT Artificial sequence generated
by phage display 66 Met His Arg Ser Asp Leu Met Ser Ala Ala Val Arg
1 5 10 67 12 PRT Artificial sequence generated by phage display 67
Met Pro Lys Leu Met Thr Met Asp Lys Ser Met Tyr 1 5 10 68 12 PRT
Artificial sequence generated by phage display 68 Asn Thr Lys Ser
Trp Ala Ala Pro Ala Pro Glu Tyr 1 5 10 69 12 PRT Artificial
sequence generated by phage display 69 Gln Gln Asn Val Ala Leu Arg
Leu Asp Trp Met Gly 1 5 10 70 12 PRT Artificial sequence generated
by phage display 70 Gln Thr Ile Thr Ser Pro Gln Met His Pro Arg Ala
1 5 10 71 12 PRT Artificial sequence generated by phage display 71
Ser Pro Thr Trp Ser Gln Ser Lys Asn Ser Asn Gln 1 5 10 72 12 PRT
Artificial sequence generated by phage display 72 Ser Arg Tyr Ile
Pro Asp Phe Ala Thr Ser Ala Pro 1 5 10 73 12 PRT Artificial
sequence generated by phage display 73 Ser Ser Pro Leu Pro Leu Ser
Met Ser Ala Pro Arg 1 5 10 74 12 PRT Artificial sequence generated
by phage display 74 Ser Ser Trp Asn Glu Ala Tyr Arg Ser Arg Ser Gly
1 5 10 75 12 PRT Artificial sequence generated by phage display 75
Ser Tyr Thr Phe His Gln Leu Pro Ser Ala His Leu 1 5 10 76 12 PRT
Artificial sequence generated by phage display 76 Thr Phe Ser Asn
Leu Gln Thr Thr Ala Gln Ala Val 1 5 10 77 12 PRT Artificial
sequence generated by phage display 77 Thr His Ile Leu Thr Lys Ser
Ala Ser Ser Tyr Ile 1 5 10 78 12 PRT Artificial sequence generated
by phage display 78 Thr His Pro Trp Ser Leu Lys Thr Thr Ser Phe Ser
1 5 10 79 12 PRT Artificial sequence generated by phage display 79
Thr Thr His Leu His Thr Asp Ser Asp Leu Gly Arg 1 5 10 80 12 PRT
Artificial sequence generated by phage display 80 Thr Thr Ile Ile
Ser Lys Asn His Ala Thr Ser Thr 1 5 10 81 12 PRT Artificial
sequence generated by phage display 81 Val Ala Pro Tyr Asn Ile Thr
Ser Pro Trp Thr Ser 1 5 10 82 12 PRT Artificial sequence generated
by phage display 82 Trp Pro His Tyr His Pro Arg Ser Thr Ile Lys Thr
1 5 10 83 12 PRT Artificial sequence generated by phage display 83
Tyr Gly Gln Asn Thr Thr Ser Pro Pro Tyr Leu Ser 1 5 10 84 12 PRT
Artificial sequence generated by phage display 84 Tyr Gln Thr Asn
Ser Tyr Asn Ala Thr Pro Ala Leu 1 5 10 85 12 PRT Artificial
sequence generated by phage display 85 Tyr Val Ser Val Gly Met Lys
Pro Ser Pro Arg Pro 1 5 10 86 12 PRT Artificial sequence generated
by phage display 86 Ala Asn Arg Ala Leu Leu Leu Asn Asp His Pro Met
1 5 10 87 12 PRT Artificial sequence generated by phage display 87
Ala Pro Ala Gly His Cys Ser Val Cys Ser Arg Ile 1 5 10 88 12 PRT
Artificial sequence generated by phage display 88 Ala Pro Asp Val
Thr Lys Val Arg Thr Lys Asp Thr 1 5 10 89 12 PRT Artificial
sequence generated by phage display 89 Asp His Trp His His Trp Cys
Asn Leu His Lys Pro 1 5 10 90 12 PRT Artificial sequence generated
by phage display 90 Glu His Arg Asn Gln Trp Cys Ile His Asp Lys Arg
1 5 10 91 12 PRT Artificial sequence generated by phage display 91
Glu Tyr Leu Ser Ala Ile Val Ala Gly Pro Trp Pro 1 5 10 92 12 PRT
Artificial sequence generated by phage display 92 Gly Pro His His
Tyr Trp Tyr His Leu Arg Leu Pro 1 5 10 93 12 PRT Artificial
sequence generated by phage display 93 His Gly Val Trp Thr Pro Trp
Met Tyr Ser Phe Ser 1 5 10 94 12 PRT Artificial sequence generated
by phage display 94 His Lys Arg His His Tyr Arg Gln Ala Cys Met His
1 5 10 95 12 PRT Artificial sequence generated by phage display 95
His Asn Trp Trp Pro Ser Trp Pro Pro Gly Pro Ser 1 5 10 96 12 PRT
Artificial sequence generated by phage display 96 His Asn Trp Tyr
His Trp Trp Met Leu Asp Asn Asn 1 5 10 97 12 PRT Artificial
sequence generated by phage display 97 His Asn Tyr Arg Ile Trp Asn
His Trp Trp Leu Ser 1 5 10 98 12 PRT Artificial sequence generated
by phage display 98 His Thr Thr Trp Pro Arg Trp Trp Ala Ser Phe Ser
1 5 10 99 12 PRT Artificial sequence generated by phage display 99
His Val Trp Glu Arg Arg Arg Thr Arg His Asn Pro 1 5 10 100 12 PRT
Artificial sequence generated by phage display 100 His Trp Arg Pro
Trp Gln His Val Ser Ser Phe Leu 1 5 10 101 12 PRT Artificial
sequence generated by phage display 101 His Trp Thr His Phe Trp Thr
Arg Thr Leu Pro His 1 5 10 102 12 PRT Artificial sequence generated
by phage display 102 His Trp Trp Thr Gly Ile Pro Thr Arg Tyr Leu
Phe 1 5 10 103 12 PRT Artificial sequence generated by phage
display 103 His Tyr Trp Trp Trp Arg Ala Met Ala Lys Gln Thr 1 5 10
104 12 PRT Artificial sequence generated by phage display 104 Lys
Pro Ile Gln Tyr Asn Asn Gly Leu Gln Ala Phe 1 5 10 105 12 PRT
Artificial sequence generated by phage display 105 Lys Thr Phe His
Ala Gly Asn Ser Asn His Leu Ala 1 5 10 106 12 PRT Artificial
sequence generated by phage display 106 Gln Ser Lys Ser His Asn Ser
Phe Leu Asn Ser Pro 1 5 10 107 12 PRT Artificial sequence generated
by phage display 107 Ser Phe Ser Pro Gln Tyr Arg Ala Pro Gly Gln
His 1 5 10 108 12 PRT Artificial sequence generated by phage
display 108 Ser Thr Asn Gln Ala Arg Phe Pro Leu His Ala Leu 1 5 10
109 12 PRT Artificial sequence generated by phage display 109 Thr
Pro Phe Leu Pro Asn Val Gly Thr Phe Ser Arg 1 5 10 110 12 PRT
Artificial sequence generated by phage display 110 Val Leu Pro His
Lys Pro Met Arg Gln Pro Val Ala 1 5 10 111 12 PRT Artificial
sequence generated by phage display 111 Tyr Ala Ser Leu Ile Asn Pro
Ile Leu Glu Pro Pro 1 5 10 112 12 PRT Artificial sequence generated
by phage display 112 Tyr His Lys Pro Phe Asn Tyr Ala Phe Pro Arg
Thr 1 5 10 113 12 PRT Artificial sequence generated by phage
display 113 Tyr Gln Gly Tyr His Arg Ser Met Pro His Gly Ser 1 5 10
114 12 PRT Artificial sequence generated by phage display 114 Ala
Pro Leu Thr Ile Thr Arg Pro Leu Trp Pro Gly 1 5 10 115 12 PRT
Artificial sequence generated by phage display 115 Ala Pro Pro Met
Ser Arg Gln Ser Phe Asp Gly Val 1 5 10 116 12 PRT Artificial
sequence generated by phage display 116 Ala Arg Phe Ile Gly Val Leu
Trp Pro Pro Thr Asn 1 5 10 117 12 PRT Artificial sequence generated
by phage display 117 Asp Pro Ala Leu Arg His Thr His His Asn Leu
Arg 1 5 10 118 12 PRT Artificial sequence generated by phage
display 118 Asp Val Ala Ile Ala Pro Lys Lys Ser Trp Val Val 1 5 10
119 12 PRT Artificial sequence generated by phage display 119 Glu
Glu Ala Asn Leu Ser Asn Val Pro Ser Trp Gly 1 5 10 120 12 PRT
Artificial sequence generated by phage display 120 Glu Gln His Pro
Arg Phe Ser Gln His Leu Leu Leu 1 5 10 121 12 PRT Artificial
sequence generated by phage display 121 Phe Asn Leu Pro Ser Lys Asn
Ser Ser Ile Ala Tyr 1 5 10 122 12 PRT Artificial sequence generated
by phage display 122 His Cys Trp Asn Gln Trp Cys Ser Arg His Gln
Thr 1 5 10 123 12 PRT Artificial sequence generated by phage
display 123 His Phe Trp Arg Pro Pro Thr Val Trp Ile Trp Pro 1 5 10
124 12 PRT Artificial sequence generated by phage display 124 His
Asn Trp Tyr Arg Trp Cys Ile Arg His Asn Asn 1 5
10 125 12 PRT Artificial sequence generated by phage display 125
His Arg Trp Tyr Arg Trp Ser Ser Ser Asn Gln Thr 1 5 10 126 12 PRT
Artificial sequence generated by phage display 126 His Ser Ser Trp
Trp Leu Ala Leu Asp Lys Pro Thr 1 5 10 127 12 PRT Artificial
sequence generated by phage display 127 His Trp Leu Pro His Asn Trp
Glu Pro Val Ala Thr 1 5 10 128 12 PRT Artificial sequence generated
by phage display 128 His Trp Ser Ala Trp Trp Ile Leu Ser Asn Gln
Ser 1 5 10 129 12 PRT Artificial sequence generated by phage
display 129 His Trp Trp Ala Trp Trp Ile Ser Ser Asn Gln Ser 1 5 10
130 12 PRT Artificial sequence generated by phage display 130 Ile
Tyr Lys Pro Gln Leu Lys Met Arg Leu Arg Leu 1 5 10 131 12 PRT
Artificial sequence generated by phage display 131 Lys Pro Pro Gln
Met Pro Leu Tyr Asn Leu Ser Ala 1 5 10 132 12 PRT Artificial
sequence generated by phage display 132 Leu Val Leu Arg Ile Ser Gln
Gly Gly Val Gly Pro 1 5 10 133 12 PRT Artificial sequence generated
by phage display 133 Met Pro His His Ala Leu Leu Gln Phe Pro Pro
Pro 1 5 10 134 12 PRT Artificial sequence generated by phage
display 134 Asn Leu Asn Ser Thr Asn Pro Asn Leu Ile Pro Phe 1 5 10
135 12 PRT Artificial sequence generated by phage display 135 Gln
Glu Ile Leu Ser Pro Pro Ser Pro Leu His Ser 1 5 10 136 12 PRT
Artificial sequence generated by phage display 136 Gln Asn Ser Ser
Met Met Leu Val Pro Trp Arg Thr 1 5 10 137 12 PRT Artificial
sequence generated by phage display 137 Ser Ile Ile Thr Thr Pro Ala
Ser Tyr Met Asp Tyr 1 5 10 138 12 PRT Artificial sequence generated
by phage display 138 Ser Leu Ser Asn Phe Lys Asn Pro Thr Gln Ala
Pro 1 5 10 139 12 PRT Artificial sequence generated by phage
display 139 Ser Asn Ile His Ser Arg Tyr Pro Leu Trp Leu Arg 1 5 10
140 12 PRT Artificial sequence generated by phage display 140 Ser
Pro Ser Pro His Ser His Asp His Leu Phe Lys 1 5 10 141 12 PRT
Artificial sequence generated by phage display 141 Ser Val Pro Val
Thr Lys Asn Pro Leu Pro Pro Arg 1 5 10 142 12 PRT Artificial
sequence generated by phage display 142 Ser Val Ser Val Gly Met Lys
Pro Ser His Arg Pro 1 5 10 143 12 PRT Artificial sequence generated
by phage display 143 Ser Tyr Trp Pro Pro Ala Pro Pro Leu Asn Thr
Phe 1 5 10 144 12 PRT Artificial sequence generated by phage
display 144 Thr Phe Asn Pro Ala Val Asn Ala Ser Ser Leu Ser 1 5 10
145 12 PRT Artificial sequence generated by phage display 145 Thr
Pro Trp Phe Gln Trp His Gln Trp Asn Leu Asn 1 5 10 146 12 PRT
Artificial sequence generated by phage display 146 Val Asn Gln Lys
Asn Ile Pro His Ala Thr His Phe 1 5 10 147 12 PRT Artificial
sequence generated by phage display 147 Tyr Gln Gly His Ala Pro Trp
Pro Ile Ile Pro His 1 5 10 148 12 PRT Artificial sequence generated
by phage display 148 Ala Leu Thr Pro Phe Tyr Gln Ala Ile Gly Ser
Arg 1 5 10 149 12 PRT Artificial sequence generated by phage
display 149 Ala Ser Ser Val Pro Leu Ser Val Arg Leu Ala His 1 5 10
150 12 PRT Artificial sequence generated by phage display 150 Asp
Phe Thr Met Gly Gln His Pro Ser Lys His Thr 1 5 10 151 12 PRT
Artificial sequence generated by phage display 151 Asp Ser Phe Pro
Thr Pro Met Arg Ala Leu Ala Ala 1 5 10 152 12 PRT Artificial
sequence generated by phage display 152 Asp Thr Arg Gln Ala Thr His
Gly Ala Tyr Arg Leu 1 5 10 153 12 PRT Artificial sequence generated
by phage display 153 Glu Thr Val Phe Phe His Thr Met Gln Ser Pro
Glu 1 5 10 154 12 PRT Artificial sequence generated by phage
display 154 Phe Ser Leu Gln Ser His Tyr Pro Phe Pro Ser Leu 1 5 10
155 12 PRT Artificial sequence generated by phage display 155 Gly
Pro Met Ser Glu Arg Ala Pro Ser Phe Thr Ile 1 5 10 156 12 PRT
Artificial sequence generated by phage display 156 His Gly Trp His
Tyr Tyr Leu Arg Thr Gln His Ser 1 5 10 157 12 PRT Artificial
sequence generated by phage display 157 His His His His Leu Arg His
Pro Phe Trp Thr His 1 5 10 158 12 PRT Artificial sequence generated
by phage display 158 His Lys Trp Pro Leu Thr Lys Leu Pro Glu Phe
Pro 1 5 10 159 12 PRT Artificial sequence generated by phage
display 159 His Leu Thr Asp Ser Thr Leu Arg Gly Leu Leu Pro 1 5 10
160 12 PRT Artificial sequence generated by phage display 160 His
Met Tyr His His Asn Ile Leu Glu Arg His Pro 1 5 10 161 12 PRT
Artificial sequence generated by phage display MISC_FEATURE
(10)..(10) Xaa can be any amino acid 161 His Asn Pro His Thr Val
Trp Thr Thr Xaa Ala His 1 5 10 162 12 PRT Artificial sequence
generated by phage display 162 His Pro His Leu Phe Thr Lys Leu Leu
Thr Tyr Lys 1 5 10 163 12 PRT Artificial sequence generated by
phage display 163 His Gln Gln Ser Tyr His Gly Ser Arg Trp Thr Pro 1
5 10 164 12 PRT Artificial sequence generated by phage display 164
Lys Ala Pro Val Ser Phe Ser Ile His Pro Ala Trp 1 5 10 165 12 PRT
Artificial sequence generated by phage display 165 Lys Thr Cys Asn
Thr Thr Arg Pro Cys Trp Asn Pro 1 5 10 166 12 PRT Artificial
sequence generated by phage display 166 Leu Asp Lys His His Leu Arg
Met Tyr Ser Leu Lys 1 5 10 167 12 PRT Artificial sequence generated
by phage display 167 Asn Met Thr Gly Ala Leu Phe Thr Pro His Ser
Phe 1 5 10 168 12 PRT Artificial sequence generated by phage
display 168 Gln Ala Asp Leu Lys Thr Pro Pro His Gln Arg Leu 1 5 10
169 12 PRT Artificial sequence generated by phage display 169 Ser
Asn Gly Pro Gln His Ser His Val Thr Ser Ser 1 5 10 170 12 PRT
Artificial sequence generated by phage display 170 Ser Ser Tyr His
His Pro Asn Phe Leu Val Ala Ala 1 5 10 171 12 PRT Artificial
sequence generated by phage display 171 Thr Leu Lys Val Ser Thr Leu
Thr Met Gly Ala Arg 1 5 10 172 12 PRT Artificial sequence generated
by phage display 172 Thr Ser Ile Ser Tyr Ser Glu Leu Thr Pro His
Thr 1 5 10 173 12 PRT Artificial sequence generated by phage
display 173 Val Glu Asp Asn Pro Pro Ala Leu Leu Val Ser Pro 1 5 10
174 12 PRT Artificial sequence generated by phage display 174 Val
Val Asn Lys Thr Leu Lys Pro Thr Pro Val Ser 1 5 10 175 12 PRT
Artificial sequence generated by phage display 175 Trp Pro His His
Pro His Ala Ala His Thr Ile Arg 1 5 10 176 12 PRT Artificial
sequence generated by phage display 176 Tyr Val Ala Met Pro Pro Ile
Tyr Pro Asn Pro Gly 1 5 10 177 12 PRT Artificial sequence generated
by phage display 177 His Asn Trp Tyr His Trp Trp Met Pro His Lys
Thr 1 5 10
* * * * *