U.S. patent application number 11/586339 was filed with the patent office on 2008-01-24 for polypeptide multilayer films and methods.
Invention is credited to Donald T. Haynie.
Application Number | 20080020402 11/586339 |
Document ID | / |
Family ID | 37946304 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080020402 |
Kind Code |
A1 |
Haynie; Donald T. |
January 24, 2008 |
Polypeptide multilayer films and methods
Abstract
Polypeptide multilayer films comprising a hydrophobic designed
polypeptide, methods of making the polypeptide multilayer films,
and methods of designing the hydrophobic polypeptide are
disclosed.
Inventors: |
Haynie; Donald T.; (New
Haven, CT) |
Correspondence
Address: |
CANTOR COLBURN, LLP
55 GRIFFIN ROAD SOUTH
BLOOMFIELD
CT
06002
US
|
Family ID: |
37946304 |
Appl. No.: |
11/586339 |
Filed: |
October 25, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60729828 |
Oct 25, 2005 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/287.2; 977/902 |
Current CPC
Class: |
A61P 37/00 20180101;
C07K 17/00 20130101; A61P 31/12 20180101; A61P 37/04 20180101; A61P
31/14 20180101; A61P 31/18 20180101; A61P 35/00 20180101; A61P
31/10 20180101; A61P 31/04 20180101; B82Y 30/00 20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2; 977/902 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12M 3/00 20060101 C12M003/00 |
Claims
1. A multilayer film comprising: a plurality of layers of
polyelectrolytes, the layers comprising alternating oppositely
charged polyelectrolytes, wherein a first layer comprises a
hydrophobic designed polypeptide, wherein the hydrophobic designed
polypeptide comprises one or more hydrophobic amino acid sequence
motifs, a length of greater than or equal to 15 amino acid
residues, and a magnitude of a net charge per residue of less than
0.4 at pH 7, the one or more hydrophobic amino acid sequence motifs
comprising length n, at least one nonpolar amino acid residue, and
a magnitude of a net charge per residue less than 0.4 but greater
than 1/n, wherein n is 5 to 15, and wherein a second layer
comprises a second layer polyelectrolyte having a charge opposite
that of the hydrophobic designed polypeptide.
2. The multilayer film of claim 1, wherein the magnitude of the net
charge per residue of the hydrophobic designed polypeptide is less
than 0.25 at pH 7.
3. The multilayer film of claim 1, wherein the second layer
polyelectrolyte comprises a hydrophilic designed polypeptide
comprising one or more hydrophilic amino acid sequence motifs,
wherein the one or more hydrophilic amino acid sequence motifs
consists of 5 to 15 amino acids and has a magnitude of a net charge
per residue of greater than 0.4, wherein the hydrophilic designed
polypeptide is at least 15 amino acids long, and has a magnitude of
a net charge per residue of greater than 0.4.
4. The multilayer film of claim 1, wherein the multilayer film is
formed on a substrate.
5. The multilayer film of claim 4, wherein the substrate is a
nitrocellulose membrane, a silicon wafer, silicone, a surface
treated with an alkylsilane, or an organic polymer lattice.
6. The multilayer film of claim 1, comprising at least 4 pairs of
alternately charged layers.
7. The multilayer film of claim 1, having a thickness of 1 nm to
100 nm.
8-17. (canceled)
18. A method for identifying a hydrophobic amino acid sequence
motif, comprising locating a starter amino acid in a first amino
acid sequence; examining a second amino acid sequence comprising
the starter amino acid and a following n-1 amino acids in the first
amino acid sequence for occurrences of positive and negative
charges at pH 7; and identifying the second amino acid sequence as
a hydrophobic amino acid sequence motif if a magnitude of a net
charge per residue of the second amino acid sequence is at least
1/n and less than 0.4; or discarding the second amino acid sequence
if the magnitude of the net charge of the second amino acid
sequence is less than 1/n or greater than or equal to 0.4, wherein
n is 5 to 15.
19. A method of designing a hydrophobic designed polypeptide,
comprising identifying a hydrophobic amino acid sequence motif
comprising n amino acids, wherein a magnitude of a net charge per
residue of the hydrophobic amino acid sequence is at least 1/n and
less than 0.4, and wherein n is 5 to 15; and covalently joining two
or more hydrophobic amino acid sequence motifs; wherein the two or
more hydrophobic amino acid sequence motifs are the same or
different.
20. The multilayer film of claim 1, wherein the hydrophobic
designed polypeptide comprises two or more hydrophobic amino
sequence motifs joined by 1-4 glycine or proline residues.
21. The multilayer film of claim 1, wherein the hydrophobic amino
acid sequence motif has a solubility at 25.degree. C. of less than
50 .mu.g/mL in water.
22. The multilayer film of claim 1, wherein the hydrophobic
designed polypeptide has a summed .alpha.-helix propensity of less
than 7.5 and a summed .beta. sheet propensity of less than 8.
23. The multilayer film of claim 1, wherein the hydrophobic amino
acid sequence motif is designed de novo.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application 60/729,828 filed Oct. 25, 2005, which is incorporated
by reference herein.
BACKGROUND
[0002] The present invention is directed to polypeptide multilayer
films, methods of making the polypeptide multilayer films, and
methods of designing the polypeptides.
[0003] Polyelectrolyte multilayer films are thin films (e.g., a few
nanometers to millimeters thick) composed of alternating layers of
oppositely charged polyelectrolytes. Such films can be formed by
layer-by-layer assembly on a suitable substrate. In electrostatic
layer-by-layer self-assembly ("ELBL"), the physical basis of
association of polyelectrolytes is electrostatics. Film buildup is
possible because the sign of the surface charge density of the film
reverses on deposition of successive layers. The general principle
of ELBL deposition of oppositely charged polyions is illustrated in
FIG. 1. The generality and relative simplicity of the ELBL film
process permits the deposition of many different types of
polyelectrolytes onto many different types of surface. Polypeptide
multilayer films are a subset of polyelectrolyte multilayer films,
comprising at least one layer comprising a charged polypeptide. A
key advantage of polypeptide multilayer films is environmental
benignity. ELBL films can also be used for encapsulation.
Applications of polypeptide films and microcapsules include, for
example, nano-reactors, biosensors, artificial cells, and drug
delivery vehicles.
[0004] The design principles for hydrophilic polypeptides suitable
for electrostatic layer-by-layer deposition in a high dielectric
constant solvent, e.g., aqueous solution, are elucidated in U.S.
Patent Publication No. 2005/0069950, incorporated herein by
reference. In brief, the suitability of a polypeptide for ELBL is
related to the net charge on the polypeptide and the length of the
polypeptide. Polypeptides having the appropriate length and charge
properties can readily be deposited to form one or more layers of a
polypeptide multilayer film. In particular, for ELBL from high
dielectric constant solvents, U.S. Patent Publication No.
2005/0069950 discloses that a hydrophilic polypeptide suitable for
ELBL from a high dielectric constant solvent preferably comprises
one or more hydrophilic amino acid sequence motifs, that is,
contiguous amino acid sequences having a length of about 5 to about
15 amino acid residues and having a linear charge density such that
the magnitude of the net charge per residue of an amino acid
sequence motif is at least 0.5. A polypeptide for ELBL can be
designed in different ways, for example, by joining a plurality of
amino acid sequence motifs to each other, either directly or by a
linker. Additional design concerns disclosed in U.S. Patent
Publication No. 2005/0069950 for designed polypeptides suitable for
ELBL include the physical structure of the polypeptides, the
physical stability of the films formed from the polypeptides, and
the biocompatibility and bioactivity of the films and the
constituent polypeptides.
[0005] U.S. Patent Publication No. 2005/0069950 discloses
polypeptide multilayer films prepared from polypeptides dissolved
in aqueous solvent and deposited into a polypeptide multilayer
film. Such peptides have a high average charge per unit length to
be soluble and monomeric in an aqueous medium. Water is a polar
solvent, having a high dielectric constant of 78.4 at 25.degree. C.
In general, polypeptides of high net charge density are soluble in
water and other polar solvents.
[0006] Hydrophobic polyelectrolytes can be used to control the
surface tension and viscosity of an aqueous solution. Such polymers
aggregate in water, and aggregation is enhanced by increasing the
ionic strength; salt screens electrostatic repulsion between
chains.
[0007] There is a need in the market for a general platform for
controlled preparation of biodegradable coatings that are
fabricated and stable in non-aqueous environments, e.g., solvents
of low dielectric constant.
SUMMARY
[0008] Disclosed herein is a multilayer film. In one embodiment, a
multilayer film comprises a plurality of layers of
polyelectrolytes, the layers comprising alternating oppositely
charged polyelectrolytes, wherein a first layer comprises a
hydrophobic designed polypeptide, wherein the hydrophobic designed
polypeptide comprises one or more hydrophobic amino acid sequence
motifs, a length of greater than or equal to 15 amino acid
residues, and a magnitude of a net charge per residue of less than
0.4 at pH 7. The one or more hydrophobic amino acid sequence motifs
comprises length n, at least one nonpolar amino acid residue, and a
magnitude of a net charge per residue less than 0.4 but greater
than 1/n, wherein n is 5 to 15. A second layer comprises a second
layer polyelectrolyte having a charge opposite that of the
hydrophobic designed polypeptide.
[0009] A method of making a multilayer film is also disclosed
herein. In one embodiment, a method of making a film comprises
depositing a first layer polyelectrolyte on a surface of a
substrate to form a first layer; and depositing a second layer
polyelectrolyte on the first layer polyelectrolyte to form a second
layer; wherein the first layer polyelectrolyte, the second layer
polyelectrolyte, or both, comprises a hydrophobic designed
polypeptide; and wherein the first layer polyelectrolyte and the
second layer polyelectrolyte have net charges of opposite polarity.
The hydrophobic designed polypeptide comprises a hydrophobic amino
acid sequence motif, a length of greater than or equal to 15 amino
acid residues, and a magnitude of a net charge per residue of less
than 0.4 at pH 7. The hydrophobic amino acid sequence motif
comprises length n, at least one nonpolar amino acid residue, and a
magnitude of a net charge per residue less than 0.4 but greater
than 1/n, wherein n is 5 to 15.
[0010] In another embodiment, a method for identifying a
hydrophobic amino acid sequence motif comprises locating a starter
amino acid in a first amino acid sequence; examining a second amino
acid sequence comprising the starter amino acid and a following n-1
amino acids in the first amino acid sequence for occurrences of
positive and negative charges at pH 7; and identifying the second
amino acid sequence as a hydrophobic amino acid sequence motif if a
magnitude of a net charge per residue of the second amino acid
sequence is at least 1/n and less than 0.4; or discarding the
second amino acid sequence if the magnitude of the net charge of
the second amino acid sequence is less than 1/n or greater than or
equal to 0.4, wherein n is 5 to 15.
[0011] In another embodiment, a method of designing a hydrophobic
designed polypeptide comprises identifying a hydrophobic amino acid
sequence motif comprising n amino acids, wherein a magnitude of a
net charge per residue of the hydrophobic amino acid sequence is at
least 1/n and less than 0.4, and wherein n is 5 to 15; and
covalently joining two or more hydrophobic amino acid sequence
motifs; wherein the two or more hydrophobic amino acid sequence
motifs are the same or different.
[0012] These and other embodiments, advantages and features of the
invention become clear when detailed description and examples are
provided in subsequent sections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a schematic of the assembly of oppositely
charged polypeptides.
DETAILED DESCRIPTION
[0014] Disclosed herein are multilayer films comprising a
hydrophobic designed polypeptide, methods of making the multilayer
films comprising a hydrophobic designed polypeptide and methods of
designing a hydrophobic designed polypeptide. Also disclosed are a
method of identifying a hydrophobic amino acid sequence motif from
known amino acid sequence data and a method of designing a
hydrophobic amino acid sequence motif de novo.
[0015] A multilayer film comprising a hydrophobic designed
polypeptide is useful for a variety of purposes, e.g., fabrication
of environmentally benign anisotropic films. Such films are useful
for a variety of purposes, e.g., coating optical devices and
waveguides, connecting micro-sized circuits, and making liquid
crystal displays. Thin films of water on the surface of antennas,
radomes or feed waveguides can cause large attenuation of
transmitted or received signals. A hydrophobic coating on such
devices can be useful in limiting such signal attenuation.
[0016] The hydrophobic designed polypeptide can be fabricated into
a multilayer film by electrostatic layer-by-layer assembly by
deposition, spraying, or another suitable method using a solution
of the hydrophobic designed polypeptide in a low dielectric
constant solvent.
[0017] As used herein, "layer" means a thickness increment, e.g.,
on a substrate for film formation, following an adsorption step.
"Multilayer" means multiple (i.e., two or more) thickness
increments. A "polyelectrolyte multilayer film" is a film
comprising two or more thickness increments of polyelectrolytes.
After deposition, the layers of a multilayer film may not remain as
discrete layers. In fact, it is possible that there is significant
intermingling of species, particularly at the interfaces of the
thickness increments.
[0018] The term "polyelectrolyte" includes polycationic and
polyanionic materials having a molecular weight of greater than
1,000. Additionally, polyelectrolytes for use in high dielectric
constant solvents comprise at least 5 charges per molecule.
Suitable polyelectrolyte materials for multilayer film assembly in
a low dielectric constant solvent include, for example, hydrophobic
polyelectrolytes. Some examples of hydrophobic polyelectrolytes are
styrene-maleic anhydride (SMA), styrene acrylates (SA), alkylated
urethane copolymers, and certain emulsion products having
chemistries related to SMA or SA.
[0019] "Amino acid" means a building block of a polypeptide. As
used herein, "amino acid" includes the 20 common naturally
occurring L-amino acids, all other natural amino acids, all
non-natural amino acids, and all amino acid mimics, e.g.,
peptoids.
[0020] "Naturally occurring amino acids," means the 20 common
naturally occurring L-amino acids, that is, glycine, alanine,
valine, leucine, isoleucine, serine, threonine, cysteine,
methionine, aspartic acid, asparagine, glutamic acid, glutamine,
arginine, lysine, histidine, phenylalanine, tyrosine, tryptophan,
and proline.
[0021] "Non-natural amino acid" means an amino acid other than any
of the 20 common naturally occurring L-amino acids. A non-natural
amino acid can have either L- or D-stereochemistry.
[0022] "Peptoid," or N-substituted glycine, means an analog of the
corresponding amino acid monomer, with the same side chain as the
corresponding amino acid but with the side chain appended to the
nitrogen atom of the amino group rather than to the .alpha.-carbons
of the residue. Consequently, the chemical linkages between
monomers in a polypeptoid are not peptide bonds, which can be
useful for limiting proteolytic digestion.
[0023] "Amino acid sequence" and "sequence" mean a contiguous
length of polypeptide chain that is at least two amino acid
residues long.
[0024] "Residue" means an amino acid in a polymer or oligomer; it
is the residue of the amino acid monomer from which the polymer was
formed. Polypeptide synthesis involves dehydration, that is, a
single water molecule is "lost" on addition of the amino acid to a
polypeptide chain.
[0025] "Amino acid sequence motif" means a contiguous amino acid
sequence comprising n amino acid residues, wherein n is 5 to
15.
[0026] "Hydrophilic amino acid sequence motif" means an amino acid
sequence motif for which the magnitude of the net charge per
residue of an amino acid sequence motif is at least 0.4;
specifically, the magnitude of the net charge per residue is at
least 0.5. As used herein, the magnitude of the net charge refers
to the absolute value of the net charge, that is, the net charge
can be positive of negative.
[0027] "Hydrophobic amino acid sequence motif" means an amino acid
sequence motif of length n comprising at least one nonpolar amino
acid residue and for which the magnitude of the net charge per
residue of the amino acid sequence motif is less than 0.4 but
greater than 1/n. In one embodiment, the magnitude of the net
charge per residue of the hydrophobic designed polypeptide is less
than 0.25 at pH 7.
[0028] "Designed polypeptide," means a polypeptide designed for use
in fabrication of a polypeptide multilayer film. A designed
polypeptide comprises one or more amino acid sequence motifs,
wherein the polypeptide is at least 15 amino acid residues long. A
practical upper limit on the length is 1,000 amino acid residues.
For a designed polypeptide comprising more than one amino acid
sequence motif, the amino acid sequence motifs are covalently
joined together, either directly or by a linker. A designed
polypeptide can comprise multiple copies of identical amino acid
sequence motifs or multiple different amino acid sequence
motifs.
[0029] "Hydrophilic designed polypeptide" means a polypeptide
comprising one or more hydrophilic amino acid sequence motifs,
wherein the polypeptide is at least 15 amino acid residues in
length and the ratio of the number of charged residues of the same
polarity minus the number of residues of the opposite polarity to
the total number of residues in the polypeptide is greater than or
equal to 0.4 at pH 7. In other words, the magnitude of the net
charge per residue of the polypeptide is greater than or equal to
0.4. In one embodiment, the ratio of the number of charged residues
of the same polarity minus the number of residues of the opposite
polarity to the total number of residues in the polypeptide is
greater than or equal to 0.5 at pH 7. In other words, the magnitude
of the net charge per residue of the polypeptide is greater than or
equal to 0.5.
[0030] "Hydrophobic designed polypeptide" means a polypeptide
comprising one or more hydrophobic amino acid sequence motifs,
wherein the polypeptide is at least 15 amino acid residues in
length and the magnitude of the net charge per residue of the
polypeptide is less than 0.4 at pH 7. In some embodiments, the
length of the hydrophobic designed polypeptide is greater than 18,
20, 25, 30, 32, or 35 amino acid residues.
[0031] A "high dielectric constant solvent" means a solvent with a
dielectric constant greater than or equal to 50 under the
conditions used to prepare the multilayer film. For example, the
dielectric constant of water at 25.degree. C. is 78.4.
[0032] A "low dielectric constant solvent" means a solvent with a
dielectric constant less than 50 under the conditions used to
prepare the multilayer film. For example, the dielectric constant
of dimethyl sulfoxide (DMSO) at 20.degree. C. is 48.9. Low
dielectric constant solvents suitable for preparation of a
hydrophobic multilayer film include DMSO, acetonitrile,
trifluoroacetic acid, N,N-dimethylformamide, ethanol, and methanol.
A mixture of solvents (for example, a mixture of acetonitrile and
toluene) resulting in a low dielectric constant for the mixture is
also considered a low dielectric constant solvent.
[0033] "Primary structure" means the contiguous linear sequence of
amino acids in a polypeptide chain, and "secondary structure" means
the more or less regular types of structure in a polypeptide chain
stabilized by non-covalent interactions, usually hydrogen bonds.
Examples of secondary structure include .alpha. helix, .beta.
sheet, and .beta. turn.
[0034] "Polypeptide multilayer film" means a film comprising one or
more designed polypeptides, wherein the designed polypeptides are
hydrophilic, hydrophobic, or a combination thereof. In some
instances, the polypeptide multilayer film comprises, in addition
to the designed polypeptide, another type of polyelectrolyte in one
or more layers, for instance a chemically modified polypeptide, a
nonbiological organic polyelectrolyte, or a polysaccharide. For
example, a polypeptide multilayer film can comprise a first layer
comprising a designed polypeptide and a second layer comprising a
polyelectrolyte have a net charge of opposite polarity to the
designed polypeptide. The second layer can be another designed
polypeptide or another polyelectrolyte. If the first layer has a
net positive charge, the second layer has a net negative charge;
and if the first layer has a net negative charge, the second layer
has a net positive charge.
[0035] "Substrate" means a solid material with a suitable surface
for adsorption of polyelectrolytes from a solution in either a high
dielectric constant solvent or a low dielectric constant solvent.
The surface of a substrate can have essentially any shape, for
example, planar, spherical, rod-shaped, etc. A substrate surface
can be regular or irregular. A substrate can be a crystal.
Substrates range in size from the nanoscale to the macro-scale.
Moreover, a substrate optionally comprises several small
sub-particles. A substrate can be made of organic material,
inorganic material, bioactive material, or a combination thereof.
Nonlimiting examples of substrates suitable for adsorption of
hydrophobic polypeptides from a low dielectric constant solvent
include silicon wafers; silicone; surfaces treated with an
alkylsilane; surfaces treated with ESSCOLAM 10.TM. and other
proprietary coatings; organic polymer lattices, e.g., polystyrene
or styrene copolymer lattices; and hydrophobic membranes, e.g.,
nitrocellulose filters.
[0036] When a substrate is disintegrated or otherwise removed
during or after film formation, it is called "a template" (for film
formation). Template particles can be dissolved in appropriate
solvents or removed by thermal treatment. If, for example,
partially cross-linked melamine-formaldehyde template particles are
used, the template can be disintegrated by mild chemical methods,
e.g., in DMSO, or by a change in pH value. After dissolution of the
template particles, hollow multilayer shells remain which are
composed of alternating polyelectrolyte layers.
[0037] The present invention provides polypeptide multilayer films,
wherein at least one layer of the film comprises a hydrophobic
designed polypeptide. Other layers comprise designed hydrophilic
polypeptides or other polycations or polyanions.
[0038] As described above, a hydrophobic designed polypeptide means
a polypeptide comprising one or more hydrophobic amino acid
sequence motifs, wherein the polypeptide is at least 15 amino acid
residues in length, and the magnitude of the net charge per residue
of the polypeptide is less than 0.4 at pH 7. For a hydrophobic
designed polypeptide comprising more than one hydrophobic amino
acid sequence motif, the hydrophobic amino acid sequence motifs are
covalently joined together, either directly or by a linker. A
preferred characteristic for a linker is that it disfavors
formation of secondary structure across two adjacent amino acid
sequence motifs. One example of a suitable linker comprises one to
four residues of amino acids known to have negligible tendencies to
form secondary structures, for example glycine or proline. In some
embodiments, the length of the hydrophobic designed polypeptide is
greater than 18, 20, 25, 30, 32 or 35 amino acid residues.
[0039] A hydrophobic designed polypeptide comprises a single given
hydrophobic amino acid sequence motif, it comprises multiple copies
of a single given hydrophobic amino acid sequence motif or it
comprises multiple hydrophobic amino acid sequence motifs, each
chosen to impart to the hydrophobic designed polypeptide particular
desired physical, chemical, or biological properties.
[0040] Optionally, a hydrophobic designed polypeptide comprises a
label for ease of detection or concentration determination.
Examples of suitable labels are tyrosine, a fluorophore, a
fluorescently labeled amino acid, a biotinylated amino acid, and
the like. The label can be placed at any suitable position in the
sequence of the hydrophobic designed polypeptide.
[0041] Further, a hydrophobic designed polypeptide optionally
comprises a moiety that can form a crosslink between two layers or
within a layer of a film comprising the polypeptide. Examples of a
crosslinking moiety are the amino acid cysteine and its peptoid
analog. The crosslinking moiety is, for example, incorporated into
the sequence of a hydrophobic amino acid sequence motif in the
hydrophobic designed polypeptide.
[0042] A hydrophobic amino acid sequence motif means an amino acid
sequence motif (i.e., a contiguous amino acid sequence comprising n
amino acid residues, wherein n is 5 to 15) comprising at least one
nonpolar amino acid residue and for which the magnitude of the net
charge per residue of the amino acid sequence motif is at least 1/n
and less than 0.4 at pH 7. Hydrophobic amino acid sequence motifs
can include polar, uncharged amino acid residues. A hydrophobic
amino acid motif can comprise a mixture of amino acid residues of
opposite charge as long as the magnitude of the net charge of the
motif meets the specified criterion.
[0043] In an embodiment, a hydrophobic amino acid sequence motif
has an average per residue hydropathy greater than 0, as calculated
with the standard values of Kyte and Doolittle (Kyte, J.;
Doolittle, R. J. Mol. Biol. 1982, 157, 105-132).
[0044] In an embodiment, a hydrophobic amino acid sequence motif
has solubility at 25.degree. C. of less than 50 .mu.g/mL in water
or other high dielectric constant solvent and solubility at
25.degree. C. greater than 50 .mu.g/mL in a solvent with a low
dielectric constant.
[0045] In one exemplary embodiment, a hydrophobic amino acid
sequence motif comprises 7 amino acid residues. The hydrophobic
amino acid sequence motif has a net charge at pH 7, either positive
or negative. The maximum magnitude of the net charge at pH 7 for a
motif size of 7 is less than 0.4*7 (<3), while the minimum
magnitude of the net charge at pH 7 for a motif size of 7 is at
least 1/7. The maximum number of charged amino acids, of any
polarity, for a motif size of 7 is about 4, and the minimum number
of nonpolar amino acids for a motif size of 7 is at least 1.
[0046] The naturally occurring amino acids having nonpolar side
chains include: alanine, cysteine, glycine, isoleucine, leucine,
methionine, phenylalanine, proline, tryptophan, tyrosine, and
valine. Nonpolar side chains consist mainly of hydrocarbon. Any
functional groups they contain are uncharged at pH 7 and are
incapable of participating in hydrogen bonding.
[0047] The naturally occurring amino acids with polar side chains
are arginine, asparagine, aspartic acid (or aspartate), glutamine,
glutamic acid (or glutamate), histidine, lysine, serine, and
threonine. Polar side chains contain functional groups that are
either charged at pH 7 or that are able to participate in hydrogen
bonding.
[0048] The naturally occurring amino acids with positively-charged
(basic) side chains at pH 7 are arginine (Arg), histidine (His),
and lysine (Lys). The naturally occurring amino acid residues with
negatively-charged (acidic) side chains at pH 7 are glutamic acid
(or glutamate) (Glu) and aspartic acid (or aspartate) (Asp).
[0049] Also provided is a method for identifying a hydrophobic
amino acid sequence motif from the genomic or proteomic information
of a specific organism, such as the human genome. For example, the
primary structure of complement C3 (gi|68766) or lactotransferrin
(gi|4505043) can be used to search for hydrophobic amino acid
sequence motifs that might be present in the amino acid
sequence.
[0050] The method comprises selecting a starter amino acid residue
in a first amino acid sequence; examining a second amino acid
sequence comprising the starter amino acid residue and the
following n-1 amino acid residues in the first amino acid sequence
for occurrences of positive and negative charges, wherein n is 5 to
15; and identifying the second amino acid sequence as a hydrophobic
amino acid sequence motif if the magnitude of the net charge of the
side chains of the n amino acid residues at pH 7 is less than 0.4*n
but greater than 1/n; or discarding the second amino acid sequence
if the magnitude of the net charge of the side chains of the n
amino acid residues at pH 7 is greater than or equal to 0.4*n. The
method can further comprise determining the net charge of the side
chains of the n amino acid residues of the second amino acid
sequence. If the second amino acid sequence is discarded, a new
search can begin at another amino acid in the first amino acid
sequence.
[0051] The invention also provides a method for de novo design of a
hydrophobic amino acid sequence motif.
[0052] De novo design of a hydrophobic amino acid sequence motif
follows essentially similar rules, except that a hydrophobic amino
acid sequence motif designed de novo is not limited to amino acids
found in nature. A length of motif n and a desired sign and
magnitude of net charge are chosen. Then, n amino acids are
selected for the amino acid sequence motif that result in the
desired sign and magnitude of charge, so that the magnitude of the
net charge of the n amino acids is at least 1/n but less than
0.4*n.
[0053] A potential advantage of de novo design of an amino acid
sequence motif is that the practitioner can select from among all
amino acids (the 20 naturally occurring ones and all non-natural
amino acids) to achieve the desired net charge, rather than being
limited to the amino acids found in a particular known protein
sequence. The larger pool of amino acids enlarges the potential
range of physical, chemical and/or biological characteristics that
can be selected in designing the sequence of the motif compared to
identification of an amino acid sequence motif in a genomic
sequence.
[0054] Another aspect of the invention provides a method of
designing a hydrophobic designed polypeptide.
[0055] The method comprises identifying a hydrophobic amino acid
sequence motif comprising n amino acids; and covalently joining two
or more hydrophobic amino acid sequence motifs, wherein the two or
more hydrophobic amino acid sequence motifs are the same or
different.
[0056] In some cases, a design concern regarding hydrophobic amino
acid sequence motifs and hydrophobic designed polypeptides is their
propensity to form secondary structures, notably .alpha. helix or
.beta. sheet. In some embodiments, it is desirable to be able to
control, e.g., minimize, secondary structure formation by the
designed polypeptides in solution in order to maximize control over
multilayer film layer formation. First, it is preferred that
sequence motifs be relatively short, that is about 5 to about 15
amino acids, because long motifs are more likely to adopt a stable
three-dimensional structure in solution. Second, a linker, such as
a glycine or proline residue, covalently joined between successive
amino acid sequence motifs in a designed polypeptide will reduce
the propensity of the polypeptide to adopt secondary structure in
solution. Glycine, for example, has a very low .alpha. helix
propensity and a very low .beta. sheet propensity, making it
energetically very unfavorable for a glycine and its neighboring
amino acids to form regular secondary structure in aqueous
solution. Third, the .alpha. helix and .beta. sheet propensity of
the designed polypeptides themselves can be minimized by selecting
amino acid sequence motifs for which the summed .alpha.-helix
propensity is less than 7.5 and the summed .beta. sheet propensity
is less than 8. "Summed" propensity means the sum of the
.alpha.-helix or .beta.-sheet propensities of all amino acids in a
motif. Amino acid sequence motifs having a somewhat higher summed
.alpha. helix propensity and/or summed .beta. sheet propensity are
suitable for ELBL, particularly when joined by linkers such as Gly
or Pro. In certain applications, the propensity of a polypeptide to
form secondary structure can be relatively high as a specific
design feature of multilayer film fabrication. The secondary
structure propensities for all 20 naturally occurring amino acids
can be calculated using the method of Chou and Fasman (see P. Chou
and G. Fasman Biochemistry 13:211 (1974), which is incorporated by
reference herein in its entirety).
[0057] Another design concern is control of the stability of
polypeptide multilayer films. Ionic bonds, hydrogen bonds, van der
Waals interactions, and hydrophobic interactions contribute to the
stability of multilayer films. In addition, covalent disulfide
bonds formed between sulfhydryl-containing amino acids in the
polypeptides within the same layer or in adjacent layers can
increase structural strength. Sulfhydryl-containing amino acids
include cysteine and homocysteine. In addition, a sulfhydryl can be
added to .beta.-amino acids such as
D,L-.beta.-amino-.beta.-cylohexyl propionic acid;
D,L-3-aminobutanoic acid; or 5-(methylthio)-3-aminopentanoic acid.
Sulfhydryl-containing amino acids can be used to "lock" (bond
together) and "unlock" layers of a multilayer polypeptide film by a
change in oxidation potential. Also, the incorporation of a
sulfhydryl-containing amino acid in a hydrophobic amino acid
sequence motif of a hydrophobic designed polypeptide enables the
use of relatively short polypeptides in multilayer film
fabrication, by virtue of intermolecular disulfide bond formation.
Hydrophobic amino acid sequence motifs containing
sulfhydryl-containing amino acids may be selected from a library of
motifs identified using the methods described below, or designed de
novo.
[0058] In one embodiment, the designed sulfhydryl-containing
polypeptides, whether synthesized chemically or produced in a host
organism, are assembled by ELBL in the presence of a reducing agent
to prevent premature disulfide bond formation. Following film
assembly, the reducing agent is removed and an oxidizing agent is
added. In the presence of the oxidizing agent disulfide bonds form
between sulfhydryls groups, thereby "locking" together the
polypeptides within layers and between layers where thiol groups
are present. Suitable reducing agents include dithiothreitol (DTT),
2-mercaptoethanol (2-ME), reduced glutathione,
tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and
combinations of more than one of these chemicals. Suitable
oxidizing agents include oxidized glutathione,
tert-butylhydroperoxide (t-BHP), thimerosal, diamide,
5,5'-dithio-bis-(2-nitro-benzoic acid) (DTNB),
4,4'-dithiodipyridine, sodium bromate, hydrogen peroxide, sodium
tetrathionate, porphyrindin, sodium orthoiodosobenzoate, and
combinations of more than one of these chemicals.
[0059] A hydrophobic designed polypeptide comprises one or more
hydrophobic amino acid sequence motifs. The same hydrophobic amino
acid sequence motif may be repeated, or different hydrophobic amino
acid sequence motifs may be joined in designing a hydrophobic
designed polypeptide for inclusion in a multilayer film. In one
embodiment, the hydrophobic amino acid sequence motifs are
covalently joined with no intervening sequence. In another
embodiment, a hydrophobic designed polypeptide comprises two or
more hydrophobic amino acid sequence motifs covalently joined by a
linker. The linker can be amino acid based, e.g., one or more amino
acid residues such as glycine or proline, or it can be any other
compound suitable for covalently linking two amino acid sequence
motifs. In one embodiment, a linker comprises 1-4 amino acid
residues, for example, 1-4 glycine and/or proline resides. The
linker comprises a suitable length or composition so that the
hydrophobic designed polypeptide is maintained at a net charge per
residue that is less than 0.4.
[0060] A hydrophobic designed polypeptide with amino acid-based
linkers is synthesized using methods well known in the art, such as
solid phase synthesis and F-moc chemistry, or heterologous
expression in bacteria following gene cloning and transformation.
Hydrophobic designed polypeptides may be synthesized by a peptide
synthesis company, for example, SynPep Corp. (Dublin, Calif.),
produced in the laboratory using a peptide synthesizer, or produced
by recombinant DNA methods. Any development of novel methods of
peptide synthesis could enhance the production of hydrophobic
designed polypeptides but would not fundamentally change peptide
design as described herein.
[0061] The invention further provides a method of making a
multilayer polypeptide film comprising a hydrophobic designed
polypeptide.
[0062] In an embodiment, the method comprises depositing a first
layer hydrophobic designed polypeptide on a surface to form a first
layer; and depositing a second layer hydrophobic designed
polypeptide on the first layer polypeptide to form a second layer;
wherein the net charge of the second layer hydrophobic designed
polypeptide is opposite in polarity to the net charge of the first
layer hydrophobic designed polypeptide; wherein each hydrophobic
designed polypeptide comprises one or more hydrophobic amino acid
sequence motifs, a length of 15 to 1000 amino acid residues, and a
magnitude of a net charge per residue of less than 0.4 at pH 7.
[0063] In another embodiment, the method comprises depositing a
first layer polypeptide on a surface to form a first layer; and
depositing a second layer polypeptide on the first layer
polypeptide to form a second layer; wherein the first layer
polypeptide, the second layer polypeptide, or both, comprises a
hydrophobic designed polypeptide; wherein the first layer
polypeptide and the second layer polypeptide have net charges of
opposite polarity; and wherein the hydrophobic designed polypeptide
comprises a hydrophobic amino acid sequence motif, a length of
greater than 15 amino acid residues, and a magnitude of a net
charge per residue of less than 0.4 at pH 7.
[0064] In another embodiment, the method comprises depositing a
first layer polyelectrolyte on a surface to form a first layer; and
depositing a second layer polyelectrolyte on the first layer
polyelectrolyte to form a second layer; wherein at least one of the
first layer polyelectrolyte and the second layer polyelectrolyte
comprises a hydrophobic designed polypeptide; wherein the first
layer polyelectrolyte and the second layer polyelectrolyte have net
charges of opposite polarity; and wherein the hydrophobic designed
polypeptide comprises a hydrophobic amino acid sequence motif, a
length of greater than or equal to amino acid residues, and a
magnitude of a net charge per residue of less than 0.4 at pH 7.
[0065] In another embodiment, the method comprises depositing a
plurality of layers of oppositely charged polyelectrolytes on a
substrate, wherein at least one layer comprises a hydrophobic
designed polypeptide.
[0066] Polyelectrolytes for deposition are dissolved in a suitable
solvent in which the polyelectrolyte has adequate solubility to
achieve an appropriate concentration for the deposition solution.
For example, for deposition of a hydrophobic designed polypeptide,
the hydrophobic designed polypeptide is dissolved in a low
dielectric constant solvent and for deposition of a hydrophilic
designed polypeptide, the hydrophilic designed polypeptide is
dissolved in a high dielectric constant solvent.
[0067] Deposition of layers of oppositely charged polypeptides onto
a substrate can be performed by any method known in the art.
Deposition of layers of oppositely charged polypeptides in solution
onto a substrate can be performed at any temperature at which a
solution comprising a polypeptide for deposition is liquid. For
example, the aqueous deposition solution of a hydrophilic designed
polypeptide can be at a temperature from about 0.degree. C. to
about 100.degree. C. for the deposition process or the deposition
solution of a hydrophobic designed polypeptide in acetonitrile can
be at a temperature from about -48.degree. C. to about 81.degree.
C.
[0068] In some embodiments, the deposition of the polypeptides is
by ELBL. Successively deposited layers have opposite net charges.
In other embodiments, the deposition on the substrate is by
successively spraying solutions of oppositely charged polypeptides.
In yet other embodiments, the deposition on the substrate is by
simultaneous spraying of solutions of oppositely charged
polypeptides, wherein the solvent of the positively charged
polypeptide solution and the solvent of the negatively charged
polypeptide solution are miscible.
[0069] ELBL is one method of making a multilayer thin film from
oppositely charged species, deposited in succession on a solid
support. The method is simple and versatile. The basic principle of
assembly, Coulombic attraction and repulsion, is far more general
than the type of adsorbing species or surface area or shape of
support. Electrostatics both drives film assembly and limits it.
Several layers of material applied in succession create a solid,
multilayer coating. Each layer can have a thickness on the order of
nanometers, enabling the design and engineering of surfaces and
interfaces at the molecular level. The layering process is
repetitive and can be automated, important for control over the
process and commercialization prospects.
[0070] In the ELBL method of forming a multilayer film, the
opposing charges of the adjacent layers provide the driving force
for assembly. It is not critical that polyelectrolytes in opposing
layers have the same net linear charge density, only that opposing
layers have opposite charges. One standard film assembly procedure
by deposition includes forming solutions of the polyions at a pH at
which they are ionized (i.e., pH 4-10), providing a substrate
bearing a surface charge, and alternating immersion of the
substrate into the charged polyelectrolyte solutions. Washing the
substrate subsequent to separation of the substrate and a
deposition solution can optionally be performed prior to exposure
of the substrate to the next deposition solution of the oppositely
charged polypeptide.
[0071] Recently, alternatives to the repetitive assembly of layers
by immersion of the substrate into deposition solutions have been
developed for the fabrication of ionic polymer films. An iterative
spraying method of film assembly has been introduced. The use of
spin-coaters has also been demonstrated. Continuous and
simultaneous spraying of polyanion and polycation solutions onto a
vertically oriented charged surface has also been shown to create a
uniform film that grows continuously with spraying time. A vertical
orientation enables continuous drainage of excess polyion and
solvent.
[0072] In some embodiments, an oppositely charged polypeptide
deposited on the substrate comprises a hydrophilic designed
polypeptide. In other embodiments, at least one of the oppositely
charged polypeptides comprises poly (L-lysine) (PLL) or poly
(L-glutamic acid) (PLGA).
[0073] The concentration of polyelectrolyte suitable for deposition
of the polyelectrolyte can readily be determined by one of ordinary
skill in the art. An exemplary concentration is 0.1 to 10 mg/mL.
Typically, the thickness of the layer produced is substantially
independent of the solution concentration of the polyelectrolyte
during deposition in the stated range. For typical non-polypeptide
polyelectrolytes such as poly(acrylic acid) and poly(allylamine
hydrochloride), typical layer thicknesses are about 3 to about 5
.ANG., depending on the ionic strength of solution. Short
polyelectrolytes typically form thinner layers than long
polyelectrolytes. Regarding film thickness, polyelectrolyte film
thickness depends on humidity as well as the number of layers and
composition of the film. For example, PLL/PLGA films 50 nm thick
shrink to 1.6 nm upon drying with nitrogen. In general, films of 1
nm to 100 nm or more in thickness can be formed depending on the
hydration state of the film and the molecular weight of the
polyelectrolytes employed in the assembly.
[0074] In addition, the number of layers required to form a stable
polyelectrolyte multilayer film will depend on the polyelectrolytes
in the film. For films comprising only low molecular weight
polypeptide layers, a film will typically have 4 or more bilayers
of oppositely charged polypeptides. For films comprising high
molecular weight polyelectrolytes such as poly(acrylic acid) and
poly(allylamine hydrochloride), films comprising a single bilayer
of oppositely charged polyelectrolyte can be stable.
[0075] The invention is further illustrated by the following
nonlimiting examples.
EXAMPLES
Example 1
Preparation of a Multilayer Film Comprising Hydrophobic Designed
Polypeptides
[0076] Two hydrophobic designed polypeptides are chemically
synthesized:
[0077] P1: (AAKAAAKG).sub.3AAKAAAKY (SEQ ID NO:1), comprises 3
copies of the hydrophobic sequence motif AAKAAAK (SEQ ID NO:2) and
1 copy of the hydrophobic sequence motif AAKAAAKY (SEQ ID NO:3)
covalently linked together by single glycine residues.
[0078] N1: (AAEAAAEG).sub.3AAEAAAEY (SEQ ID NO:4), comprises 3
copies of the hydrophobic sequence motif AAEAAAE (SEQ ID NO:5) and
1 copy of the hydrophobic sequence motif AAEAAAEY (SEQ ID NO:6)
covalently linked together by single glycine residues.
[0079] A multilayer film comprising the two hydrophobic designed
polypeptides is formed at 25.degree. C. by depositing a layer of P1
dissolved in acetonitrile onto a substrate, e.g., a nitrocellulose
membrane, having a relatively low surface charge density. The
substrate is immersed in the P1 solution for 15 minutes to permit
adsorption of P1 to the substrate, and then removed and rinsed in
acetonitrile. The substrate is then immersed in a solution of N1 in
acetonitrile to permit adsorption of N1 to the substrate. Following
adsorption, the substrate is removed from the N1 solution and
rinsed with acetonitrile.
[0080] Additional layers are deposited in like manner. A total of
five layers of P1 and 5 layers of N1 are alternately deposited.
Example 2
Preparation of a Multilayer Film Comprising Hydrophobic Designed
Polypeptides and Hydrophilic Designed Polypeptides
[0081] Four designed polypeptides are chemically synthesized, two
hydrophobic and two hydrophilic.
[0082] The two hydrophobic designed polypeptides are P1 and N1,
described above in Example 1.
[0083] The two hydrophilic designed polypeptide are the
following:
[0084] P2: (KAKAKAKG).sub.3KAKAKAKY (SEQ ID NO:7), comprises 3
copies of the hydrophilic sequence motif KAKAKAK (SEQ ID NO:8) and
1 copy of the hydrophilic sequence motif KAKAKAKY (SEQ ID NO:9)
covalently linked together by single glycine residues.
[0085] N2: (EAEAEAEG).sub.3EAEAEAEY (SEQ ID NO:10), comprises 3
copies of the hydrophilic sequence motif EAEAEAE (SEQ ID NO:11) and
1 copy of the hydrophilic sequence motif EAEAEAEY (SEQ ID NO:12)
covalently linked together by single glycine residues.
[0086] A multilayer film comprising the two hydrophobic designed
polypeptides and the two hydrophilic designed polypeptides is
formed at 25.degree. C. as follows. P1 is dissolved in a low
dielectric constant solvent, e.g., acetonitrile. N1 is dissolved in
a low dielectric constant solvent. A layer of P1 is deposited onto
a substrate having a relatively low surface charge density, e.g., a
nitrocellulose membrane. The substrate is immersed in the P1
solution for 15 minutes to permit adsorption of P1 to the
substrate, and then removed and rinsed in acetonitrile to remove
loosely bound polypeptide. The substrate is then immersed in a
solution of N1 in acetonitrile to permit adsorption of N1 to the
substrate. Following adsorption, the substrate is removed from the
N1 solution and rinsed with acetonitrile to remove loosely bound
polypeptide.
[0087] Additional layers of hydrophobic peptide are deposited in
like manner.
[0088] A hydrophobic film is stable in a hydrophilic solvent
because hydrophobic polypeptides are insoluble in a hydrophilic
solvent.
[0089] A hydrophilic film is stable in a hydrophobic solvent
because hydrophilic peptides are insoluble in a hydrophobic
solvent.
[0090] Layers of hydrophilic designed polypeptides are deposited on
top of the hydrophobic film at 25.degree. C. as follows. P2 is
dissolved in a hydrophilic solvent, e.g., a mixture of water and
acetonitrile. N2 is dissolved in a hydrophilic solvent. A layer of
P2 is deposited onto the multilayer film surface. The film is
immersed in the P2 solution for 15 minutes to permit adsorption of
P2 to the surface, and then removed and rinsed in the mixture of
water and acetonitrile to remove loosely bound peptide. The
substrate is then immersed in a solution of N2 in the mixture of
water and acetonitrile to permit adsorption of N2 to the surface.
Following adsorption, the film is removed from the N2 solution and
rinsed with the mixture of water and acetonitrile to remove loosely
bound peptide.
[0091] An increasingly hydrophilic film can be built by depositing
increasingly hydrophilic polypeptides from an increasingly
hydrophilic solvent, e.g., an increasing percentage of water in a
water/methanol mixture.
[0092] An increasingly hydrophobic film can be built by depositing
increasingly hydrophobic polypeptides from an increasingly
hydrophobic solvent, e.g., a decreasing percentage of water in a
water/methanol mixture.
[0093] The use of the terms "a" and "an" and "the" and similar
referents (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms first, second etc. as used herein are not meant to denote any
particular ordering, but simply for convenience to denote a
plurality of, for example, layers. The terms "comprising",
"having", "including", and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to")
unless otherwise noted. Recitation of ranges of values are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. The
endpoints of all ranges are included within the range and
independently combinable. All methods described herein can be
performed in a suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all
examples, or exemplary language (e.g., "such as"), is intended
merely to better illustrate the invention and does not pose a
limitation on the scope of the invention unless otherwise claimed.
No language in the specification should be construed as indicating
any non-claimed element as essential to the practice of the
invention as used herein.
[0094] While the invention has been described with reference to an
exemplary embodiment, it will be understood by those skilled in the
art that various changes may be made and equivalents may be
substituted for elements thereof without departing from the scope
of the invention. In addition, many modifications may be made to
adapt a particular situation or material to the teachings of the
invention without departing from the essential scope thereof.
Therefore, it is intended that the invention not be limited to the
particular embodiment disclosed as the best mode contemplated for
carrying out this invention, but that the invention will include
all embodiments falling within the scope of the appended claims.
Any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
12 1 32 PRT Artificial Synthetic construct 1 Ala Ala Lys Ala Ala
Ala Lys Gly Ala Ala Lys Ala Ala Ala Lys Gly 1 5 10 15 Ala Ala Lys
Ala Ala Ala Lys Gly Ala Ala Lys Ala Ala Ala Lys Tyr 20 25 30 2 7
PRT Artificial Synthetic construct 2 Ala Ala Lys Ala Ala Ala Lys 1
5 3 8 PRT Artificial Synthetic construct 3 Ala Ala Lys Ala Ala Ala
Lys Tyr 1 5 4 32 PRT Artificial Synthetic construct 4 Ala Ala Glu
Ala Ala Ala Glu Gly Ala Ala Glu Ala Ala Ala Glu Gly 1 5 10 15 Ala
Ala Glu Ala Ala Ala Glu Gly Ala Ala Glu Ala Ala Ala Glu Tyr 20 25
30 5 7 PRT Artificial Synthetic construct 5 Ala Ala Glu Ala Ala Ala
Glu 1 5 6 8 PRT Artificial Synthetic construct 6 Ala Ala Glu Ala
Ala Ala Glu Tyr 1 5 7 32 PRT Artificial Synthetic construct 7 Lys
Ala Lys Ala Lys Ala Lys Gly Lys Ala Lys Ala Lys Ala Lys Gly 1 5 10
15 Lys Ala Lys Ala Lys Ala Lys Gly Lys Ala Lys Ala Lys Ala Lys Tyr
20 25 30 8 7 PRT Artificial Synthetic construct 8 Lys Ala Lys Ala
Lys Ala Lys 1 5 9 8 PRT Artificial Synthetic construct 9 Lys Ala
Lys Ala Lys Ala Lys Tyr 1 5 10 32 PRT Artificial Synthetic
construct 10 Glu Ala Glu Ala Glu Ala Glu Gly Glu Ala Glu Ala Glu
Ala Glu Gly 1 5 10 15 Glu Ala Glu Ala Glu Ala Glu Gly Glu Ala Glu
Ala Glu Ala Glu Tyr 20 25 30 11 7 PRT Artificial Synthetic
construct 11 Glu Ala Glu Ala Glu Ala Glu 1 5 12 8 PRT Artificial
Synthetic construct 12 Glu Ala Glu Ala Glu Ala Glu Tyr 1 5
* * * * *