U.S. patent application number 11/372901 was filed with the patent office on 2006-07-13 for method for design of polypeptides for nanofabrication of multilayer films, coatings, and microcapsules.
Invention is credited to Donald T. Haynie.
Application Number | 20060155482 11/372901 |
Document ID | / |
Family ID | 34375754 |
Filed Date | 2006-07-13 |
United States Patent
Application |
20060155482 |
Kind Code |
A1 |
Haynie; Donald T. |
July 13, 2006 |
Method for design of polypeptides for nanofabrication of multilayer
films, coatings, and microcapsules
Abstract
A method for designing polypeptides suitable, for example, for
the nanofabrication of thin films, coatings, and microcapsules by
ELBL for applications in biomedicine and other fields is described.
In one embodiment, the method comprises identifying an amino acid
sequence motif comprising n+1 amino acids, wherein a balance of
charges of the same charge in the amino acid sequence motif is
greater than or equal to one half of n; and covalently joining two
or more amino acid sequence motifs having the same net charge;
wherein the two or more amino acid sequence motifs are the same or
different.
Inventors: |
Haynie; Donald T.; (Ruston,
LA) |
Correspondence
Address: |
Karen A. LeCuyer;Cantor Colburn, LLP
55 Griffin Road South
Bloomfield
CT
06002
US
|
Family ID: |
34375754 |
Appl. No.: |
11/372901 |
Filed: |
March 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10652364 |
Aug 29, 2003 |
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11372901 |
Mar 10, 2006 |
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Current U.S.
Class: |
702/19 |
Current CPC
Class: |
G01N 33/6803
20130101 |
Class at
Publication: |
702/019 |
International
Class: |
G06F 19/00 20060101
G06F019/00 |
Claims
1. A method of designing a peptide, comprising identifying an amino
acid sequence motif comprising n+1 amino acids', wherein a balance
of charges of the same charge in the amino acid sequence motif is
greater than or equal to one half of n; and covalently joining two
or more amino acid sequence motifs having the same net charge;
wherein the two or more amino acid sequence motifs are the same or
different.
2. The method of claim 1, wherein the two amino acid sequence
motifs are covalently joined by a glycine linker, or a proline
linker.
3. The method of claim 1, wherein n is 4 to 12.
4. The method of claim 1, wherein the amino acid sequence motif
comprises a cysteine residue.
5. The method of claim 1, wherein the amino acid sequence motif
comprises no amino acids having a charge opposite the overall
charge of the amino acid sequence motif.
6. The method of claim 1, wherein the peptide is biocompatible.
7. The method of claim 1, wherein the peptide comprises at least
one non-natural amino acid.
8. The method of claim 1, wherein the polypeptide polypeptide
comprises a functional domain, and wherein the functional domain
functions in ligand binding, in vivo targeting, biosensing, or
biocatalysis.
9. The method of claim 1, wherein identifying 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 amino acids in the first amino acid sequence for
occurrences of positive and negative charges; determining the
second amino acid sequence as the amino acid sequence motif if the
number of charges having the same charge in the second amino acid
sequence is greater than or equal to one half of n; or discarding
the second amino acid sequence if the number of charges having the
same charge in the second amino acid sequence is less than one half
of n.
10. The method of claim 9, further comprising locating a second
starter amino acid in the first amino acid sequence, examining a
third amino acid sequence comprising the second starter amino acid
and a following n amino acids in the first amino acid sequence for
occurrences of positive and negative charges; determining the third
amino acid sequence as the amino acid sequence motif if a number of
charges of the same charge in the third amino acid sequence is
greater than or equal to one half of n, and the number of charges
of the opposite charge is zero; or discarding the third amino acid
sequence if both positive and negative charges occur in the
sequence.
11. The method of claim 10, wherein the two amino acid sequence
motifs are covalently joined by a glycine linker, or a proline
linker.
12. The method of claim 9, wherein n of the amino acid sequence
motif is 4 to 12.
13. The method of claim 9, wherein the amino acid sequence motif
comprises a cysteine residue.
14. The method of claim 9, wherein the first amino acid sequence is
found in human proteome data.
15. A method for identifying an amino acid sequence motif for
incorporation into a peptide, 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 amino
acids in the first amino acid sequence for occurrences of positive
and negative charges; identifying the second amino acid sequence as
the amino acid sequence motif if the number of charges having the
same charge in the second amino acid sequence is greater than or
equal to one half of n; or discarding the second amino acid
sequence if both positive and negative charges occur in the
sequence.
16. The method of claim 15, wherein n of the amino acid sequence
motif is 4 to 12.
17. The method of claim 15, wherein the amino acid sequence motif
comprises a cysteine residue.
18. The method of claim 15, wherein the first amino acid sequence
is found in human proteome data.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a division of U.S. Nonprovisional
application Ser. No. 10/652,364 filed Aug. 29, 2003, which is
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the fabrication of
ultrathin multilayered films on suitable surfaces by electrostatic
layer-by-layer self assembly ("ELBL"). More specifically, the
present invention relates to a method for designing polypeptides
for the nanofabrication of thin films, coatings, and microcapsules
by ELBL for applications in biomedicine and other fields.
[0004] 2. Description of Related Art
[0005] ELBL is an established technique in which ultrathin films
are assembled by alternating the adsorption of oppositely-charged
polyelectrolytes. The process is based on the reversal of the
surface charge of the film after the deposition of each layer. FIG.
1 shows a schematic diagram of the general ELBL process: films of
oppositely charged polyions (cationic polyions 10 and anionic
polyions 11) are assembled in successive layers on a
negatively-charged planar surface 12; the surface charge is
reversed after the deposition of each layer. This process is
repeated until a film of desired thickness is formed. The physical
basis of association is electrostatics-gravitation and nuclear
forces play effectively no role. Because of the generality and
relative simplicity of the process, ELBL allows for the deposition
of many different types of materials onto many different types of
surface. There is, therefore, a vast number of possible useful
combinations of materials and surfaces. For a general discussion of
ELBL, including its history, see Yuri Lvov, "Electrostatic
Layer-by-Layer Assembly of Proteins and Polyions" in Protein
Architecture: Interfacial Molecular Assembly and Immobilization
Biotechnology, Y. Lvov & H. Mohwald eds. (New York: Marcel
Dekker, 1999), pp. 125-167, which is incorporated herein by
reference in its entirety.
[0006] ELBL has recently become a focus area in the field of
nanotechnology because it can be used to fabricate films
substantially less than 1 micron in thickness. Moreover, ELBL
permits exceptional control over the film fabrication process,
enabling the use of nanoscale materials and permitting nanoscale
structural modifications. Because each layer has a thickness on the
order of a few nanometers or less, depending on the type of
material used and the specific adsorption process, multilayer
assemblies of precisely repeatable thickness can be formed.
[0007] A number of synthetic polyelectrolytes have been employed in
ELBL applications, including sodium poly(styrene sulfonate)
("PSS"), poly(allylamine hydrochloride) ("PAH"),
poly(diallyldimethylammonium chloride) ("PDDA"),
poly(acrylamide-co-diallyldimethylammonium chloride),
poly(ethyleneimine) ("PEI"), poly(acrylic acid) ("PAA"),
poly(anetholesulfonic acid), poly(vinyl sulfate) ("PVS"), and
poly(vinylsulfonic acid). Such materials, however, are not
generally useful for biomedical applications because they are
antigenic or toxic.
[0008] Proteins, being polymers with side chains having ionizable
groups, can be used in ELBL for various applications, including
biomedical ones. Examples of proteins that have been used in ELBL
include cytochrome c, hen egg white lysozyme, immunoglobulin G,
myoglobin, hemoglobin, and serum albumin (ibid.). There are,
however, difficulties with using proteins for this purpose. These
include limited control over multilayer structure (because the
surface of the protein is highly irregular and proteins will not
ordinarily adsorb on a surface in a regular pattern), restrictions
on pH due to the pH-dependence of protein solubility and structural
stability, lack of biocompatibility when using exogenous proteins,
and the cost of scaling up production if the gene has not been
cloned; unless the protein were identical in a readily available
source, e.g. a cow, the protein would have to be obtained from the
organism in which it was intended for use, making the cost of
large-scale production of the protein prohibitive.
[0009] By contrast polypeptides, which are generally smaller and
less complex than proteins, constitute an excellent class of
material for ELBL assembly, and polypeptide film structures formed
by ELBL will be useful in a broad range of applications. The
present invention provides a method for designing polypeptides for
the nanofabrication of thin films, coatings, and microcapsules by
ELBL. Polypeptides designed using the method of the present
invention should exhibit several useful properties, including,
without limitation, completely determined primary structure,
minimal secondary structure in aqueous solution, monodispersity,
completely controlled net charge per unit length, ability to form
cross-links on demand, ability to reverse cross-link formation,
ability to form more organized thin films than is possible with
proteins, and relatively inexpensive large-scale production cost
(assuming gene design, synthesis, cloning, and host expression in
E. Coli or yeast, or peptide synthesis).
[0010] Polypeptides designed using the method of the present
invention have been shown useful for ELBL of thin film structures
with targeted or possible applications in biomedical technology,
food technology, and environmental technology. Such polypeptides
could be used, for example, to fabricate artificial red blood
cells, drug delivery devices, and antimicrobial films.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention provides a novel method for
identifying "sequence motifs" of a defined length and net charge at
neutral pH in amino acid sequence information for use in ELBL, and
recording a desired number of the motifs. The method comprises the
steps of: (a) Obtaining an amino acid sequence for a peptide or a
protein from a particular organism; (b) Locating a starter amino
acid in the amino acid sequence; (c) Examining the starter amino
acid and the following n amino acids to determine the number of
charged amino acids having a polarity opposite the certain
polarity; (d) If the number of the charged amino acids having a
polarity opposite the certain polarity is one or more, continuing
the method at step g; (e) Examining the starter amino acid and the
following n amino acids to determine the number of charged amino
acids having the certain polarity; (f) If the number of charged
amino acids having the certain polarity is equal to or greater than
x, recording the amino acid sequence motif consisting of the
starter amino acid and the following n amino acids; (g) Locating
another starter amino acid in the amino acid sequence; and (h)
Repeating the method beginning at step c until the desired number
of amino acid sequence motifs have been identified or all of the
amino acids in the amino acid sequence have been used as the
starter amino acid in step c; wherein x is greater than or equal to
approximately one-half of n.
[0012] The present invention also provides a novel method for
designing a polypeptide for use in ELBL, comprising the steps of:
(a) Identifying and recording one or more amino acid sequence
motifs having a net charge of a certain polarity using the steps
mentioned in the preceding paragraph and (b) Joining a plurality of
said recorded amino acid sequence motifs to form a polypeptide.
[0013] The present invention also provides a novel method for
designing a polypeptide for use in ELBL comprising the following
steps: (a) Designing de novo a plurality of amino acid sequence
motifs, wherein said amino acid sequence motifs consist of n amino
acids, at least x of which are positively charged and none is
negatively charged, or at least x of which are negatively charged
and none is positively charged, wherein x is greater than or equal
to approximately one-half of n; and (b) Joining said plurality of
said amino acid sequence motifs. The amino acid sequence motifs can
comprise the 20 usual amino acids or non-natural amino acids, and
the amino acids can be either left-handed (L-amino acids) or right
handed (D-amino acids).
[0014] The present invention also provides a thin film, the film
comprising a plurality of layers of polypeptides, the layers of
polypeptides having alternating charges, wherein the polypeptides
comprise at least one amino acid sequence motif consisting of n
amino acids, at least x of which are positively charged and none is
negatively charged, or at least x of which are negatively charged
and none is positively charged, wherein x is greater than or equal
to approximately one-half of n. The motifs in these polypeptides
may be selected using either of the methods described above.
[0015] The present invention also provides a novel process for
using cysteine and other sulphydryl-containing amino acid types to
"lock" and "unlock" the layers of polypeptide ELBL films. This
process enables the films to remain stable at extremes of pH,
giving greater control over the mechanical stability and diffusive
properties of films nanofabricated from designed polypeptides and
increasing their utility in a broad range of applications.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0016] FIG. 1 is a schematic diagram of the general ELBL
process.
[0017] FIG. 2 is a graph of the cumulative secondary structure
propensities of the amino acid sequence motifs identified in human
amino acid sequence information using the method of the present
invention, compared with the distribution of structure propensities
of 10.sup.5 random amino acid sequences.
[0018] FIG. 3(a) shows adsorption data as monitored by the quartz
crystal microbalance technique ("QCM") for a combination of amino
acid sequences designed according to the present invention.
[0019] FIG. 3(b) shows a comparison of adsorption data as monitored
by QCM for different combinations of amino acid sequences designed
according to the present invention.
[0020] FIG. 3(c) shows a graph of adsorbed mass in nanograms versus
layer number for amino acid sequences designed and fabricated
according to the present invention.
[0021] FIG. 4(a) illustrates intra-layer disulfide bonds according
to the cysteine locking method of the present invention.
[0022] FIG. 4(b) illustrates inter-layer disulfide bonds according
to the cysteine locking method of the present invention.
[0023] FIG. 4(c) illustrates the oxidation and reduction of
disulfide bonds in microcapsules fabricated from polypeptides
designed according to the method of the present invention.
[0024] FIG. 5 is a schematic of the selection process of the
present invention used to identify in existing amino acid sequence
information amino acid sequence motifs having suitable
electrostatic properties for ELBL.
[0025] FIG. 6 shows the number of non-redundant sequence motifs
identified in available human amino acid sequence data.
[0026] FIG. 7 shows the ELBL adsorption of poly-L-glutamate and
poly-L-lysine from an aqueous medium as a function of ionic
strength.
[0027] FIG. 8 shows the adsorption of polypeptides designed
according to the method of the present invention for experiments to
probe the effect of disulfide bond formation.
[0028] FIG. 9 shows the percentage of material remaining during
thin film disassembly at acidic pH as discussed with reference to
FIG. 8.
[0029] FIG. 10 shows the percentage of material lost during the
acidic pH disassembly step of an experiment involving de
novo-designed polypeptides containing cysteine.
[0030] FIG. 11(a) illustrates the role of solution structure of
peptides on film assembly, showing how the assembly behavior of
poly-L-glutamate and poly-L-lysine depends on pH. QCM resonant
frequency is plotted against adsorption layer. The average
molecular mass of poly-L-glutamate was 84,600 Da, while that of
poly-L-lysine was 84,000 Da. The numbers refer to pH values. E=Glu,
K=Lys. The peptide concentration used for assembly was 2 mg/mL.
[0031] FIG. 11(b) illustrates the role of solution structure of
peptides on film assembly, showing how the solution structure of
poly-L-glutamate and poly-L-lysine depends on pH. Mean molar
residue ellipticity is plotted as a function of pH. The peptide
concentration was 0.05 mg/mL.
[0032] FIG. 12 shows adsorption data for polyelectrolytes of
different lengths, illustrating that long polyelectrolytes adsorb
better than short ones.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Explanations of Terms
[0034] For convenience in the ensuing description, the following
explanations of terms are adopted. However, these explanations are
intended to be exemplary only. They are not intended to limit the
terms as they are described or referred to throughout the
specification. Rather, these explanations are meant to include any
additional aspects and/or examples of the terms as described and
claimed herein.
[0035] As used herein, "biocompatibility" means causing no adverse
health effect upon ingestion, contact with the skin, or
introduction to the bloodstream.
[0036] As used herein, "immune response" means the response of the
human immune system to the presence of a substance in the
bloodstream. An immune response can be characterized in a number of
ways, for example, by an increase in the bloodstream of the number
of antibodies that recognize a certain antigen. (Antibodies are
proteins made by the immune system, and an antigen is an entity
that generates an immune response.) The human body fights infection
and inhibits reinfection by increasing the number of antibodies in
the bloodstream. The specific immune response depends somewhat on
the individual, though general patterns of response are the
norm.
[0037] As used herein, "epitope" means the structure of a protein
that is recognized by an antibody. Ordinarily an epitope will be on
the surface of a protein. A "continuous epitope" is one that
involves several amino acids in a row, not one that involves amino
acid residues that happen to be in contact in a folded protein.
[0038] As used herein, "sequence motif" and "motif" mean an amino
acid sequence of a given number of residues identified using the
method of the current invention. In a preferred embodiment, the
number of residues is 7.
[0039] As used herein, "amino acid sequence" and "sequence" mean
any length of polypeptide chain that is at least two amino residues
long.
[0040] As used herein, "residue" means an amino acid in a polymer;
it is the residue of the amino acid monomer from which the polymer
was formed. Polypeptide synthesis involves dehydration--a single
water molecule is "lost" on addition of the amino acid to a
polypeptide chain.
[0041] As used herein, "designed polypeptide" means a polypeptide
designed using the method of the present invention, and the terms
"peptide" and "polypeptide" are used interchangeably.
[0042] As used herein, "primary structure" means the linear
sequence of amino acids in a polypeptide chain, and "secondary
structure" means the more or less regular types of structure
stabilized by non-covalent interactions, usually hydrogen
bonds--examples include .alpha.-helix, .beta.-sheet, and
.beta.-turn.
[0043] As used herein, "amino acid" is not limited to the 20
naturally occurring amino acids; the term also refers to D-amino
acids, L-amino acids, and non-natural amino acids, as the context
permits.
[0044] As used herein, "non-natural amino acids" means amino acids
other than the 20 naturally occurring ones.
[0045] The following three-letter abbreviations are used herein for
the 20 usual amino acids: TABLE-US-00001 Ala = alanine Cys =
cysteine Asp = aspartic acid Glu = glutamic acid Phe =
phenylalanine Gly = glycine His = histidine Ile = isoleucine Lys =
lysine Leu = leucine Met = methionine Asn = asparagine Pro =
proline Gln = glutamine Arg = arginine Ser = serine Thr = threonine
Val = valine Trp = tryptophan Tyr = tyrosine
[0046] A. Description of the Invention
[0047] The present invention provides a method for designing
polypeptides for the nanofabrication by ELBL of thin films,
coatings, and microcapsules for applications in biomedicine and
other fields. The method involves 5 primary design concerns: (1)
the electrostatic properties of the polypeptides; (2) the physical
structure of the polypeptides; (3) the physical stability of the
films formed from the polypeptides; (4) the biocompatibility of the
polypeptides and films; and (5) the bioactivity of the polypeptides
and films. The first design concern, electrostatics, is perhaps the
most important because it is the basis of ELBL. Without suitable
charge properties, a polypeptide will not be soluble in aqueous
solution and cannot be used for the ELBL nanofabrication of films.
We have devised a novel process for identifying in amino acid
sequence information amino acid sequence motifs having
electrostatic properties suitable for ELBL.
[0048] The secondary structure of the polypeptides used for ELBL is
also important, because the physical properties of the film,
including its stability, will depend on how the solution structure
of the peptide translates into its structure in the film. FIG. 11
illustrates how the solution structure of certain polypeptides
correlates with film assembly. Panel (a) shows how the assembly
behavior of poly-L-glutamate and poly-L-lysine depends on pH. It is
clear that the .alpha.-helix conformation correlates with a greater
extent of deposited material than the .beta.-sheet conformation.
The precise molecular interpretation of this behavior remains to be
elucidated. Panel (b) shows how the solution structure of these
peptides depends on pH. At pH 4.2 poly-L-glutamate is largely
.alpha.-helical, as is poly-L-lysine at pH 10.5. Both polypeptides
are in a largely unstructured coil-like conformation at pH 7.3.
[0049] The remaining concerns relate to the applications of the
polypeptide films. In practicing the invention, more or less weight
will be placed on these other concerns depending on the design
requirements of a particular application.
[0050] By using the selection process of the present invention to
identify in amino acid sequence information amino acid sequence
motifs having suitable charge characteristics, and using the other
design concerns to select particular motifs, one can design
polypeptides suitable for the ELBL fabrication of nano-organized
films for applications in biomedicine and other fields.
Alternatively, one can use the method of the present invention to
design polypeptides de novo for use in ELBL. The approach to de
novo design is essentially the same as identifying motifs in
existing amino acid sequence information, except that each residue
in an amino acid sequence motif is selected by the practitioner
rather than an entire motif being identified in the genomic or
proteomic information of a specific organism. It must be emphasized
that the fundamental polypeptide design principles adduced in the
present invention are independent of whether the amino acids
involved are the 20 naturally-occurring ones, non-natural amino
acids, or some novel combination of these, in the case of de novo
polypeptide design. Further, both D-amino acids and L-amino acids
could be used.
[0051] The design concerns of the present invention are discussed
in more detail below.
[0052] 1. Electrostatics
[0053] We have devised a novel process for identifying in amino
acid sequence information amino acid sequence motifs having
electrostatic properties suitable for ELBL. Using this process, we
have identified 88,315 non-redundant amino acid sequence motifs in
human proteome data--the translation of the portion of the genome
that encodes all known proteins in the human body. This information
is publicly available at the National Center for Biotechnology
Information's ("NCBI") Web site:
<http://www.ncbi.nlm.nih.gov>, among other places. Such
information is constantly being updated as the human genome is
further analyzed. As the amount of such information increases, the
number of amino acid sequence motifs that could be identified in
human sequence information by the selection process of the present
invention as having suitable electrostatic properties for ELBL will
also increase. The same is true for any organism. Accepted
biochemical and physics principles, as well as the experimental
results described below, indicate that the identified sequence
motifs will be useful for the design of polypeptides for the
nanofabrication of ELBL structures.
[0054] The key selection criterion is the average charge per unit
length at neutral pH (pH 7, close to the pH of human blood). In
addition, there are several structural preferences. First, it is
preferred that each amino acid sequence motif consist of only 7
residues.
[0055] a. Total Number of Residues in the Motif
[0056] The motif length of 7 was chosen in an effort to optimize
biocompatibility, physical structure, and the number of
non-redundant sequence motifs in available amino acid sequence
data.
[0057] As discussed below, it is preferred that at least half of
the amino acid residues in each sequence motif be charged.
Moreover, it is preferred that all of the charged residues in each
motif be of the same charge. These requirements ensure that each
motif will be sufficiently soluble in aqueous solvent and have
sufficient charge at neutral pH to be useful for ELBL. Because only
a relatively small percentage of amino acid types are charged, as
the length of a given amino acid sequence increases, the odds
decrease that the sequence will have a sufficient percentage of
appropriately charged amino acids for ELBL. 4 charged amino acids
is the preferred minimum for a motif size of 7, because fewer than
4 charges yields substantially decreased peptide solubility and
decreased control over ELBL.
[0058] Regarding biocompatibility (discussed further below), each
identified sequence motif is long enough at 7 residues to
constitute a continuous epitope (relevant to the possible immune
response of an organism into which a designed peptide might be
introduced), but not so long as to correspond substantially to
residues both on the surface of a protein and in its interior; the
charge requirements help to ensure that the sequence motif occurs
on the surface of the folded protein; a charged residue cannot be
formed in the core of a folded protein. By contrast, a very short
motif could appear to the body to be a random sequence, or one not
specifically "self," and therefore elicit an immune response.
Although the ideal length of a peptide for generating antibodies is
a point of some dispute, most peptide antigens range in length from
12 to 16 residues. Peptides that are 9 residues or shorter can be
effective antigens; peptides longer than 12 to 16 amino acids may
contain multiple epitopes (Angeletti, R. H. (1999) Design of Useful
Peptide Antigens, J. Biomol. Tech. 10:2-10, which is hereby
incorporated by reference in its entirety). Thus, to minimize
antigenicity one would prefer a peptide shorter than 12 and, better
yet, shorter than 9 residues.
[0059] The preferred motifs should not be too long for another
reason: to minimize secondary structure formation. Secondary
structure decreases control of the physical structure of the
polypeptides (see below) and the films made from them.
[0060] Furthermore, the maximum number of non-redundant motifs is
found when the number of residues in each motif is 7. FIG. 6 shows
the number of non-redundant sequence motifs in available human
amino acid sequence information. The greatest number of positive
motifs is for a 5-residue length, while the greatest number of
negative motifs is for a 7-residue length. The greatest number of
positive and negative motifs is about the same for 5 and 7. Thus, a
motif length of 7 residues would appear to maximize the number of
non-redundant motifs.
[0061] For all of the above reasons, 7 residues is the preferred
length of motif to optimize polypeptide design for ELBL.
Nevertheless, it is possible that in some cases either slightly
shorter or slightly longer motifs will work equally as well. For
example, motifs 5 or 6 residues long may be employed, and motifs on
the order of 8 to 15 residues in length could also be useful.
[0062] b. Number of Charged Residues
[0063] Second, it is preferred that at least 4 positively-charged
(basic) amino acids (Arg, His, or Lys) or at least 4
negatively-charged (acidic) amino acids (Glu or Asp) are present in
each 7-residue motif at neutral pH. Combinations of positive and
negative charges are disfavored in an effort to ensure a
sufficiently high charge density at neutral pH. It is possible,
however, that a motif containing both positive and negative amino
acids could be useful for ELBL. For example, a slightly longer
motif, say of 9 residues, could have 6 positively charged amino
acids and 1 negatively charged amino acid. It is the balance of
charge that is important--the overall peptide must be either
sufficiently positively charged or sufficiently negatively charged
at neutral pH. Preferred embodiments of the motifs, however, will
contain only Glu or Asp or only Arg, His, or Lys as the charged
amino acids (although other non-charged amino acids could, and
ordinarily do, form part of the motifs), unless non-natural amino
acids are admitted as acidic or basic amino acids.
[0064] FIG. 5 is a flow chart showing the steps involved in the
selection process for identifying amino acid sequences having
suitable electrostatic properties. It is assumed that only the 20
usual amino acids are involved. If searching for negatively-charged
motifs, the process begins by locating an amino acid in the
sequence data. This amino acid will be called the "starter amino
acid" because it is the starting point for the analysis of the
surrounding amino acids (i.e., it will begin the motif). Next, the
starter amino acid and the following 6 residues are examined for
occurrences of Arg, His, or Lys. If one or more Arg, His, or Lys is
located in these 7 amino acids, the process is begun anew at
another starter amino acid. If no Arg, His, or Lys is found, the 7
amino acids are examined to determine the number of occurrences of
Glu and/or Asp. If there are at least 4 occurrences of Glu and/or
Asp in the 7 residues, the sequence motif is cataloged. The
selection process is essentially the same for positively charged
amino acids, except that Glu and Asp are replaced by Arg, His, and
Lys, and Arg, His, and Lys are replaced by Glu and Asp,
respectively. Obviously, one could also begin the method at the
beginning of the amino acid sequence (amino terminus) and proceed
to the end (carboxyl terminus), or, alternatively, one could begin
at a random point and work through all of the amino acids in the
sequence, randomly or systematically in either direction. Moreover,
one could use the method to identify motifs in sequence information
containing non-natural amino acids, for example if codes were used
for each non-natural amino acid type. In such a case, one would
search for non-natural acidic or basic amino acids instead of Glu
and Asp, and Arg, Lys, and His, respectively.
[0065] The remaining design concerns, namely, physical structure,
physical stability, biocompatibility, and biofunctionality, deal
primarily with the particular application for which the designed
polypeptides will be used. As noted above, more or less weight will
be placed on these concerns during the design process, depending on
the desired peptide properties for a particular application.
[0066] 2. Physical Structure
[0067] A design concern regarding the amino acid sequence motifs is
their propensity to form secondary structures, notably
.alpha.-helix or .beta.-sheet. We have sought in several ways to
control, notably minimize, secondary structure formation of
designed polypeptides in an aqueous medium in order to maximize
control over thin film layer formation. First, it is preferred that
the sequence motifs be relatively short, because long motifs are
more likely to adopt a stable three-dimensional structure in
solution. Second, we place a glycine residue between each motif in
preferred embodiments of the polypeptide designs. Glycine 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. Proline has similar properties in some
respects and could be used as an alternative to glycine to join
motifs. Third, we have sought to minimize the .alpha.-helix and
.beta.-sheet propensity of the designed polypeptides themselves by
focusing on 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.) It is
possible, however, that amino acid sequences having a somewhat
higher summed .alpha.-helix propensity and/or summed .beta.-sheet
propensity would be suitable for ELBL under some circumstances, as
the Gly (or Pro) residues between motifs will play a key role in
inhibiting stable secondary structure formation in the designed
polypeptide. In fact, it may be desirable in certain applications
for the propensity of a polypeptide to form secondary structure to
be relatively high, as a specific design feature of thin film
fabrication; the necessary electrostatic charge requirements for
ELBL must still be met, as discussed above.
[0068] In order to be able to select amino acid sequences with
desired secondary structure propensities, we first calculated the
secondary structure propensities for all 20 amino acids 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) using structural information from more than 1,800
high-resolution X-ray crystallographic structures (1,334 containing
.alpha.-helices and 1,221 containing .beta.-strands). Structures
were selected from the Protein Data Bank (a publicly-accessible
repository of protein structures) based on: (a) method of structure
determination (X-ray diffraction); (b) resolution (better than 2.0
.ANG.)-"resolution" in this context refers to the minimum size of a
structure one can resolve, as in the Rayleigh criterion; and (c)
structural diversity (less than 50% sequence identity between the
protein crystallographic structures used to compute the helix and
sheet propensities of the various amino acids). The rationale was
to choose high resolution structures determined by the most
reliable methodology and not to bias the propensity calculation by
having similar structures. Next, for comparison 100,000
non-redundant random sequences were produced using a random number
generator in a personal computer. We then calculated the secondary
structure propensities for the 88,315 amino acid sequences
identified using the selection process described in part VII(B)(1)
above (59,385 non-redundant basic sequence motifs and 28,930
non-redundant acidic sequence motifs). The propensities for the
random sequences were then compared to the propensities of the
selected sequences. FIG. 2 shows the distribution of secondary
structure formation propensities in these sequence motifs. The
rectangle in FIG. 2 highlights the sequence motifs we have
identified as least likely to form secondary structure on the basis
of secondary structure propensities.
[0069] 3. Physical Stability
[0070] Another design concern is control of the stability of the
polypeptide ELBL films. Ionic bonds, hydrogen bonds, van der Waals
interactions, and hydrophobic interactions provide some, albeit
relatively limited, stability to ELBL films. By contrast, covalent
disulfide bonds could provide exceptional structural strength. We
have devised a novel process for using cysteine (or some other type
of sulfhydryl-containing amino acid) to "lock" and "unlock"
adjacent layers of polypeptide ELBL film. This process enables a
polypeptide nanofabricated film to remain stable at extremes of pH,
giving greater control over its mechanical stability and diffusive
properties (for discussions of porosity of multilayer films made of
non-polypeptide polyelectrolytes, see Caruso, F., Niikura, K.,
Furlong, N. and Okahata (1997) Langmuir 13:3427 and Caruso, F.,
Furlong, N., Ariga, K., Ichinose, I., and Kunitake, T. (1998)
Langmuir 14:4559, both of which are incorporated herein by
reference in their entireties). Also, the incorporation of cysteine
(or some other type of sulfhydryl-containing amino acid) in a
sequence motif of a designed polypeptide enables the use of
relatively short peptides in thin film fabrication, by virtue of
intermolecular disulfide bond formation. Without cysteine, such
peptides would not generally yield sufficiently stable films (see
FIG. 12, discussed below). Thus, our novel use of cysteine will
obviate the need to produce expensive long versions of the designed
polypeptides in a substantial percentage of possible applications.
This will be particularly advantageous in situations where the thin
film is to be fabricated over material to be encapsulated, for
example a small crystal of a drug, a small spherical hemoglobin
crystal, or a solution containing hemoglobin.
[0071] For applications in which the physical stability of the
films is important, amino acid sequence motifs containing cysteine
(or some other type of sulfhydryl-containing amino acid) may be
selected from the library of motifs identified using the methods
discussed above, or designed de novo using the principles described
above. Polypeptides can then be designed and fabricated based on
the selected or designed amino acid sequence motifs. Once the
polypeptides have been synthesized chemically or produced in a host
organism, ELBL assembly of cysteine-containing peptides is done in
the presence of a reducing agent, to prevent premature disulfide
bond formation. Following assembly, the reducing agent is removed
and an oxidizing agent is added. In the presence of the oxidizing
agent disulfide bonds form between cysteine residues, thereby
"locking" together the polypeptide layers that contain them.
[0072] This "locking" method may be further illustrated using the
following specific example of microcapsule fabrication. First,
designed polypeptides containing cysteine are used to form
multilayers by ELBL on a suitably charged spherical surface,
normally in aqueous solution at neutral pH and in the presence of
dithiothreitol ("DTT"), a reducing agent. Next, DTT is removed by
filtration, diffusion, or some other similar method known in the
art, causing cystine to form from pairs of cysteine side chains and
thereby stabilizing the film. If the peptide multilayers are
constructed on a core particle containing the materials one wishes
to encapsulate, for instance a crystalline material, the
fabrication process is complete and the core particle can
thereafter be made to dissolve in the encapsulated environment, for
example by a change of pH. If, however, the multilayers are
constructed on a "dummy" core particle, the core must be removed.
In the case of melamine formaldehyde particles ("MF"), for example,
the core is ordinarily dissolved by decreasing the pH--dissolution
is acid-catalyzed. Following dissolution of the core, the pH of
solution is adjusted to 4, where partial charge on the peptide
polyanions makes the microcapsules semi-permeable (compare Lvov et
al. (2001) Nano Letters 1:125, which is hereby incorporated herein
in its entirety). Next, 10 mM DTT is added to the microcapsule
solution to reduce cystine to cysteine. The microcapsules may then
be "loaded" by transferring them to a concentrated solution of the
material to be encapsulated, for example a protein (ibid.). The
protein enters the microcapsules by moving down its concentration
gradient. The encapsulated protein is "locked in" by removal of
reductant and addition of oxidant, thereby promoting the
reformation of disulfide bonds.
[0073] A schematic of the cysteine "locking" and "unlocking" method
of the present invention is shown in FIG. 4. Cysteine can form both
intra- and inter-molecular disulfide bonds. Further, disulfide
bonds can be formed between molecules in the same layer or adjacent
layers, depending on the location of cysteine-containing peptides
in the film. Referring to FIG. 4(a), basic polypeptides 2 are
linked by disulfide bonds 3 in all layers in which the basic
peptides contain cysteine. The acidic peptides of the intervening
layer (represented in the figure by a translucent layer 4) do not
contain cysteine. However, alternating layers continue to attract
each other electrostatically, if the acidic and basic side chains
are charged at the pH of the surrounding environment. Referring to
FIG. 4(b), disulfide bonds are shown between layers. Such
structures will form when both the acidic and basic polypeptides
(i.e., alternating polypeptide layers) used for ELBL contain
cysteine and the procedure used has been suitable for disulfide
bond formation. Referring to FIG. 4(c), reduction and oxidation
reactions are used to regulate the release of encapsulated
compounds 5 by breaking and forming disulfide bonds 3,
respectively, and thereby regulating the diffusion of particles
through the capsule wall.
[0074] The cysteine "locking" and "unlocking" is a novel way of
regulating the structural integrity and permeability of ELBL films.
It is known in the art that glutaraldehyde can be used to
cross-link proteins, and this chemical could therefore be used to
stabilize polypeptide films. Glutaraldehyde cross-linking, however,
is irreversible. In contrast, the cysteine "locking" and
"unlocking" method of the present invention is reversible and,
therefore, offers better control over structure formation and,
importantly, use of the films and capsules that can be fabricated
using the present invention. Blood is an oxidizing environment.
Thus, in certain biomedical applications, for example artificial
red blood cells or drug delivery systems fabricated from designed
polypeptides, exposing Cys-crosslinked polypeptide film to the
blood or some other oxidizing environment after the formation of
disulfide bonds is not expected to cause those bonds to be broken.
Finally, it should also be noted that applications involving
non-natural amino acids would replace Cys with some other
sulfhydryl-containing amino acid type. For example, a sulfhydryl
could be added to .beta.-amino acids such as
D,L-.beta.-amino-.alpha.-cylohexyl propionic acid;
D,L-3-aminobutanoic acid; or 5-(methylthio)-3-aminopentanoic acid
(see http://www.synthatex.com).
[0075] 4. Biocompatibility
[0076] Biocompatibility is a major design concern in biomedical
applications. In such applications, the practitioner of the present
invention will aim to identify genomic or proteomic information
that will yield "immune inert" polypeptides, particularly if the
fabricated or coated object will make contact with circulating
blood. For purposes of the present invention, it is preferred that
the selection process discussed in Part VII(B)(1) above be used to
analyze the amino acid sequences of blood proteins. This will
maximize the odds of minimizing the immune response of an
organism.
[0077] Computer algorithms exist for predicting the antigenicity of
an amino acid sequence. Such methods, however, are known in the art
to be semi-reliable at best. In the present invention, the sequence
motifs identified using the selection method discussed above in
Part VII(B)(1) are highly polar. The motifs must, therefore, occur
on the surface of the native state of the proteins of which they
are part of the sequence. The "surface" is that part of a folded
protein that is in contact with the solvent or inaccessible to the
solvent solely because of the granular nature of water. The
"interior" is that part of a folded protein that is inaccessible to
solvent for any other reason. A folded globular soluble protein is
like an organic crystal, the interior being as densely packed as in
a crystal lattice and the exterior being in contact with the
solvent, water. Because of their charge properties, the polypeptide
sequence motifs identified using the method of the present
invention must occur mostly, if not exclusively, on the surface of
a protein. Thus, all of the sequence motifs identified in human
blood proteins using the selection process of the current invention
are effectively always in contact with the immune system while the
protein is in the blood. This holds for all conformations of the
protein that might become populated in the bloodstream, including
denatured states, because it is highly energetically unfavorable to
transfer a charge from an aqueous medium to one of low dielectric
(as occurs in a protein interior). Accepted biochemical principles
indicate, therefore, that the polypeptides designed from blood
proteins using the method of the present invention will either not
illicit an immune response or will elicit a minimal immune
response. For the same reasons, polypeptides designed using the
method of the present invention should be biocompatible. All
sequence motifs identified from genomic data using the selection
process of the current invention, not only those in blood proteins,
should be biocompatible, though the extent of immune response or
any other type of biological response may well depend on specific
details of a sequence motif. (Because the polypeptide sequences on
which the motifs are based actually occur in the organism for which
the film as been fabricated, this approach will, at least in
principle, work equally well for any type of organism. For
instance, the approach may be of significant value to veterinary
science.) Both immune response and biocompatibility are important
regarding the use of the designed peptides in biomedical
applications, including, without limitation, the manufacture of
artificial red blood cells, drug delivery systems, or polypeptides
for fabrication of biocompatible films to coat implants for
short-term or long-term introduction into an organism.
[0078] 5. Bioactivity
[0079] In some applications of polypeptide thin films, coatings, or
microcapsules, it may be desirable to modify the design of the
polypeptides to include a functional domain for use in some layer
of the structure, often the outermost. A functional domain in this
context is an independently thermostable region of a protein that
has specific biofunctionality (e.g. binding phosphotyrosine). It is
well known in the art that such biofunctionality may be integrated
with other functionalities in a multi-domain protein, as for
example in the protein tensin, which encompasses a phosphotyrosine
binding domain and a protein tyrosine phosphatase domain. The
inclusion of such a domain in a designed polypeptide could function
in a number of ways, including without limitation specific ligand
binding, targeting in vivo, biosensing, or biocatalysis.
[0080] B. Uses for Polypeptides Designed Using the Method of the
Present Invention
[0081] As noted above, polypeptides of suitable design are
excellent materials for ELBL, and polypeptide film structures
formed using ELBL will be useful in a large number of different
types of applications. Polypeptides designed using the method of
the present invention have been shown to be useful for ELBL of film
structures for possible applications in biomedical technology, food
technology, and environmental technology. For example, such
polypeptides could be used to fabricate artificial red blood
cells.
[0082] 1. Artificial Red Blood Cells
[0083] A number of different approaches have been taken to red
blood cell substitute development. One approach involves the use of
perfluorocarbons. Perfluorocarbon emulsions contain synthetic
fluorinated hydrocarbons capable of binding oxygen and delivering
it to tissues. This approach however, increases
reticulo-endothelial cell blockage. The perfluorocarbons can become
trapped in the reticulo-endothelial system, which may result in
adverse consequences.
[0084] Another approach focuses on antigen camouflaging, which
involves coating red blood cells with a biocompatible polymer
called polyethylene glycol (PEG). The PEG molecules form permanent
covalent bonds on the surface of the cell. The coating effectively
hides the antigenic molecules on the surface of the red blood
cells, so that the blood recipient's antibodies do not recognize
the cells as foreign. For example, the immune system of a normal
person who has type A blood will naturally have antibodies that
recognize antigens on the surface of type B red blood cells,
leading to cell destruction. The attachment of PEG to the surface
of a type B red blood cell "camouflages" the surface of the cell,
so that its surface antigens can no longer be recognized by the
immune system and the antigenically-foreign red blood cells will
not be destroyed as quickly (see Pargaonkar, N. A., G. Sharma, and
K. K. Vistakula. (2001) "Artificial Blood: Current Research
Report," which is hereby incorporated by reference in its
entirety).
[0085] A number of diseases, including thalassemia, that require
repeated blood transfusions are often complicated by the
development of antibodies to "minor" red cell antigens. This
"allosensitization" can render these patients almost impossible to
transfuse, rendering the situation life-threatening. In in vitro
testing, the PEG-modified red cells appear not to trigger
allosensitization and may also be useful in clinical situations
where allosensitization has already occurred (see Scott, M. D. et
al. (1997) "Chemical camouflage of antigenic determinants: Stealth
erythrocytes," Proc. Natl. Acad. Sci. USA. 94 (14): 7566-7571,
which is hereby incorporated by reference in its entirety).
[0086] Other approaches involve purified hemoglobin. Unmodified
cell-free hemoglobin has known limitations. These include oxygen
affinity that is too high for effective tissue oxygenation, a
half-life within the intravascular space that is too short to be
clinically useful, and a tendency to undergo dissociation into
dimers with resultant renal tubular damage and toxicity. Because of
these limitations, hemoglobin used to make a cell-free red blood
cell substitute must be modified. A number of modification
techniques have been developed. Hemoglobin can be cross-linked (a
covalent bond between two molecules is made by chemical
modification) and polymerized using reagents such as
glutaraldehyde. Such modifications result in a product that has a
higher P.sub.50 (partial pressure of oxygen at which 50% of all
oxygen-binding sites are occupied) than that of normal hemoglobin,
and an increase in the plasma half-life of up to 30 hours. The
source of the hemoglobin for this purpose can be human (outdated
donated blood), bovine, or human recombinant. The solution of
modified hemoglobin is prepared from highly purified hemoglobin and
taken through various biochemical processes, to eliminate
phospholipids, endotoxins, and viral contaminants (see Nester, T.
and Simpson, M (2000) "Transfusion medicine update," Blood
Substitutes, which is hereby incorporated by reference in its
entirety). Biopure Corporation (Cambridge, Mass.) has been using
modified hemoglobin for their product, Hemopure.
[0087] The main potential adverse effect of modified hemoglobin
solutions is an increase in systemic and pulmonary vascular
resistance that may lead to a decrease in cardiac index. Decreases
in the cardiac index may impair optimum oxygen delivery and
outweigh the advantage of an oxygen-carrying solution (see Kasper
S. M. et al. (1998) "The effects of increased doses of bovine
hemoglobin on hemodynamics and oxygen transport in patients
undergoing preoperative hemodilution for elective abdominal aortic
surgery," Anesth. Analg. 87: 284-91, which is hereby incorporated
by reference in its entirety). One study has examined the utility
of these solutions in the acute resuscitation phase of unstable
trauma patients. Design of the study, however, was poor, and any
role of the solutions in influencing ultimate patient outcome was
unclear (see Koenigsberg D. et al. (1999) "The efficacy trial of
diaspirin cross-linked hemoglobin in the treatment of severe
traumatic hemorrhagic shock," Acad. Emerg. Med. 6: 379-80, which is
hereby incorporated by reference in its entirety).
[0088] Many of the problems of cell-free hemoglobin can be overcome
by encapsulating it with an artificial membrane. Liposomes are
being used to encapsulate hemoglobin for use as a blood substitute.
The approach is technically challenging because not only must the
hemoglobin be prepared, it must be encapsulated in relatively high
concentration and yield. The final products must be sterile and the
liposomes must be relatively uniform in size.
[0089] Encapsulated hemoglobin has several advantages over
cell-free hemoglobin. Firstly, the artificial cell membrane
protects hemoglobin from degradative and oxidative forces in the
plasma. Secondly, the membrane protects the vascular endothelium
from toxic effects of hemoglobin. These relate to heme loss, the
production O.sub.2 free radicals and vasoconstrictor effects of NO
binding. Thirdly, encapsulation greatly increases the circulating
persistence of the hemoglobin. Moreover, encapsulated hemoglobin
can be freeze-dried for convenient storage.
[0090] Liposomal encapsulation involves phospholipids, as in cell
membranes. One major problem associated with liposomal
encapsulation, however, is that it is very difficult to regulate
the average size and distribution of liposomes. Another is that
unlike red blood cells, liposomes are often not very stable, as
they ordinarily lack an organized cytoskeleton. Yet another problem
is that liposomes often consist of multiple layers of phospholipid.
(A recent review of blood substitute development is presented in
Stowell et al. (2001) Progress in the development of RBC
substitutes, Transfusion 41:287-299, which is hereby incorporated
by reference in its entirety. See also Chang, T. 1998 "Modified
hemoglobin-based blood substitutes: cross linked, recombinant and
encapsulated hemoglobin," Artificial Cell 74 Suppl 2:233-41, which
is hereby incorporated by reference in its entirety.)
[0091] Red blood cell substitutes employing polypeptides designed
using the method of the present invention should offer several
advantages over approaches to the development of red blood cell
substitutes known in the art, including, without limitation,
superior oxygen and carbon dioxide binding functionality, lower
production cost (large-scale and therefore low-cost production is
possible because bacteria can be used to mass-produce the peptides
and because peptide ELBL can be automated), the possibility of
using suitable preparations of hemoglobin as a template for ELBL,
polypeptide biodegradability, the immune "inertness" of designed
polypeptides based on blood protein structure, and the structural
stability exhibited by designed polypeptide films, which exceeds
that of liposomes. Polypeptide ELBL assembly yields semi-porous
films, minimizing the amount of material required for fabricating a
means of encapsulation and enabling glucose, oxygen, carbon
dioxide, and various metabolites to diffuse as freely through the
films as a lipid bilayer. In contrast, other polymers potentially
suitable for this purpose have undesirable side effects--for
example, polylactide degrades into lactic acid, the substance that
causes muscle cramps, and poly (styrene sulfonate) is not
biocompatible.
[0092] Microcapsules could be formed of designed polypeptides to
encapsulate hemoglobin to serve as a red blood cell substitute.
Hemoglobin polypeptide microcapsules could also be engineered to
incorporate enzymes, including superoxide dismutase, catalase, and
methemoglobin reductase, which are ordinarily important for red
blood cell function. Moreover, the nanofabricated microcapsules can
predictably be dehydrated, suggesting that artificial red blood
cells made as described herein could be dehydrated, without loss of
function, particularly because hemoglobin can be lyophilized (i.e.,
freeze-dried) and reconstituted without loss of function, and
polyion films are stable to dehydration. This will be important for
long-term storage, transport of blood substitutes, battlefield
applications (particularly in remote locations), and space
exploration.
[0093] Polypeptides designed using the method of the present
invention could also be used for drug delivery.
[0094] 2. Drug Delivery
[0095] Micron-sized "cores" of a suitable therapeutic material in
"crystalline" form can be encapsulated by designed polypeptides,
and the resulting microcapsules could be used for drug delivery.
The core must be insoluble under some conditions, for instance high
pH or low temperature, and soluble under the conditions where
controlled release will occur. The surface charge on the crystals
can be determined by .zeta.-potential measurements (used to
determine the charge in electrostatic units on colloidal particles
in a liquid medium). The rate at which microcapsule contents are
released from the interior of the microcapsule to the surrounding
environment will depend on a number of factors, including the
thickness of the encapsulating shell, the polypeptides used in the
shell, the presence of disulfide bonds, the extent of cross-linking
of peptides, temperature, ionic strength, and the method used to
assemble the peptides. Generally, the thicker the capsule, the
longer the release time--the principle resembles that of gel
filtration chromatography.
[0096] Some work has been done on sustained release from ELBL
microcapsules (see Antipov, A., Sukhorukov, G. B., Donath, E., and
Mohwald, H. (2001) J. Phys. Chem. B, 105:2281-2284 and Freemantle,
M. (2002) Polyelectrolyte multilayers, Chem. Eng. News, 80: 44-48,
both of which are incorporated herein by reference in their
entireties). Polyelectrolytes that have been used are PSS, PAH,
PAA, PVS, PEI, and PDDA.
[0097] Polypeptides designed using the method of the present
invention should offer a number of advantages in the context of
drug delivery, including without limitation control over the
physical, chemical, and biological characteristics of the
microcapsule; the ability to make capsules with a diameter of less
than 1 mm, making the capsules suitable for injection; low
likelihood of eliciting an immune response; generally high
biocompatibility of capsules; control over the diffusive properties
of the microcapsules by varying the thickness of the layers and
using cysteine, as discussed below; the ability to target specific
locations by modification of the microcapsule surface using the
highly reactive sulfhydryl groups in cysteine (as is well known in
the art, free sulfhydryl groups, free amino groups, and free
carboxyl groups are sites to which molecules for specific targeting
could be attached), or by incorporation of a specific functional
domain in the design of the polypeptide; and the ability of
microstructures to be taken up by cells using either endocytosis or
pinocytosis.
[0098] Polypeptides designed using the method of the present
invention could also be used for antimicrobial coatings.
[0099] 3. Antimicrobial Coatings
[0100] The method of the present invention could be used to
manufacture films encompassing antimicrobial peptides. For example,
one suitable sequence might be Histatin 5, which occurs in humans:
TABLE-US-00002 (SEQ ID NO: 8) Asp Ser His Ala Lys Arg His His Gly
Tyr Lys Arg Lys His Glu Lys His His Ser His Arg Gly Tyr
The preponderance of positive charge at slightly basic pH makes
this sequence quite suitable for ELBL. It could be appended to a
peptide designed using the method of the present invention,
resulting in an antimicrobial peptide suitable for use in ELBL.
This peptide could be used as an anti-biofouling coating. For
instance, the peptide could be used to form a coating on devices
used for implantation.
[0101] There are also a number of other areas in which polypeptides
designed using the method of the present invention could be
useful.
[0102] 4. Other Uses
[0103] Other possible uses for peptides designed using the method
of the present invention include without limitation food covers,
wraps, and separation layers; food casings, pouches, bags, and
labels; food coatings; food ingredient microcapsules; drug
coatings, capsules, and microcapsules; disposable food service
items (plates, cups, cutlery); trash bags; water-soluble bags for
fertilizer and pesticides; microcapsules for fertilizer and
pesticides; agricultural mulches; paper coatings; loose-fill
packaging; disposable medical products (e.g. gloves and gowns); and
disposable diapers.
[0104] C. Fabrication
[0105] Once amino acid sequence motifs have been selected from
those identified using the method discussed in Part VII(B)(1) above
or designed de novo, the designed polypeptide is synthesized using
methods well known in the art, such as solid phase synthesis and
F-moc chemistry or heterologous expression following gene cloning
and transformation. 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 methods.
[0106] In one embodiment, a designed polypeptide consists of
individual amino acid sequence motifs joined in tandem. The same
motif may be repeated, or different motifs may be joined in
designing a polypeptide for ELBL. Moreover, functional domains may
be included, as discussed above. Other amino acids than glycine
could be used to link the sequence motifs, and amino acids other
than the 20 usual ones could be included in the motifs themselves,
depending on the properties desired of the polypeptide. Other
properties could likewise be specified by design requirements,
using methods known in the art. For example, proline could be
included for turn formation, glycine for chain flexibility, and
histidine for pH-sensitive charge properties near neutral pH.
"Hydrophobic" amino acids could also be included-hydrophobic
residue content could play a role in assembly behavior and
contribute to layer stability in a way resembling the hydrophobic
stabilization of globular proteins.
[0107] It is preferred that fabricated polypeptides be at least 15
amino acids long, although it is more preferred that the fabricated
polypeptides be at least 32 amino acids long. The reason for this
is that the entropy loss per molecule is so thermodynamically
unfavorable for short polymers that adsorption to an
oppositely-charged surface is inhibited, even if the polypeptide
has a charge per unit length of 1; long polyelectrolytes adsorb
better than short ones. This is illustrated in FIG. 12. The average
molecule masses of the peptides utilized for the length-dependence
studies were 1,500-3,000 Da (poly-L-glutamate, "small"), 3,800 Da
(poly-L-lysine, "small"), 17,000 Da (poly-L-glutamate, "medium"),
48,100 Da (poly-L-lysine, "medium"), 50,300 Da (poly-L-glutamate,
"large"), and 222,400 Da (poly-L-lysine, "large"). The data shown
in FIG. 12 clearly indicate that ELBL depends on length of peptide.
Inclusion of Cys enables the use of relatively small peptides for
ELBL, because the sulfhydryl group can be used to form disulfide
crosslinks between polypeptides.
[0108] D. Experiments
1. EXAMPLE 1
Design of Polypeptides Based on Human Blood Protein Sequences and
their Use in Polypeptide Film Fabrication
[0109] For this work, amino acid sequences were selected using the
process described in Part VII(B)(1) above to identify sequence
motifs in the primary structure of human blood proteins: Complement
C3 (gi|68766) was the source of the anionic sequence motifs, and
lactotransferrin (gi|4505043) the source of the cationic sequence
motifs. As discussed above, blood protein sequences were used to
minimize the immune response of patients into whom devices
involving the polypeptides might be introduced (including, e.g.
artificial red blood cells). In principle, this approach should be
applicable for any organism having an immune system; it is not
limited to humans. Polypeptides were synthesized by SynPep Corp.
(Dublin, Calif.). The polypeptide sequences were: TABLE-US-00003
(SEQ ID NO: 2) Tyr Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu
Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys
Gln Asp (SEQ ID NO: 1) Tyr Arg Arg Arg Arg Ser Val Gln Gly Arg Arg
Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln Gly Arg Arg Arg
Arg Ser Val Gln (SEQ ID NO: 4) Tyr Glu Glu Asp Glu Cys Gln Asp Gly
Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Gly Glu
Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu
Asp Glu Cys Gln Asp (SEQ ID NO: 3) Tyr Arg Arg Arg Arg Ser Val Gln
Gly Arg Arg Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln Gly
Arg Arg Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln Gly Arg
Arg Arg Arg Ser Val Gln
[0110] The amino acid residues are represented by the three-letter
code given above. One glycine was introduced between each 7-residue
motif to inhibit secondary structure formation. Glycine was
selected for this purpose because it allows the greatest
variability in combination of dihedral angles (see Ramachandran, G.
N. and Saisekharan, V. (1968), Adv. Protein Chemistry, 23:283,
which is incorporated by reference herein in its entirety) and has
a low helix propensity (0.677) and low sheet propensity (0.766).
Alternatively, proline could be substituted for glycine between
motifs on the basis of calculated structure propensities.
Additionally, a single tyrosine was included at the N-terminus of
each peptide for concentration determination by UV absorption at
280 nm.
[0111] The polypeptides were named SN1 (SEQ ID NO: 2), SP2 (SEQ ID
NO: 1), LN3 (SEQ ID NO: 4), and LP4 (SEQ ID NO: 3), respectively,
meaning short negative, short positive, long negative, and long
positive. These sequences are quite different from polylysine
(commonly used in the art as a polycation) and polyglutamate
(commonly used in the art as a polyanion) which, though available
commercially and inexpensive, have a high .alpha.-helix propensity
under conditions of mild pH and, crucially, are immunoreactive. The
calculated charge per unit length on the designed peptides at
neutral pH is 0.5 electrostatic units for SP and LP and 0.6
electrostatic units for SN and LN. The positive peptides are
somewhat more hydrophobic than the negative ones, owing to the
presence of valine and the long hydrocarbon side chain of arginine.
(As mentioned above, hydrophobic interactions between polypeptide
layers could stabilize films to some extent.) The lengths are
consistent with published studies showing that polyions must have
greater than 20 charged groups (i.e. aspartic acid and glutamic
acid; lysine, arginine, and histidine) to be suitable for ELBL (see
Kabanov, V. and Zezin, A. (1984) Pure Appl. Chem. 56:343 and
Kabanov, V. (1994) Polym. Sci. 36:143, both of which are
incorporated by reference herein in their entireties).
[0112] a. Experimental Demonstration
[0113] i. Materials
[0114] QCM electrodes (USI-System, Japan) coated with evaporated
silver had a surface area of 0.16.+-.0.01 cm.sup.2 on each side, a
resonant frequency of 9 MHz (AT-cut), and a long-term stability of
.+-.2 Hz. The polypeptide molecular weight was verified by
electrospray mass spectrometry. Peptide purity was greater than
70%. The polypeptide buffer was 10 mM sodium phosphate or 10 mM
Tris-HCl, 1 mM DTT, 0.1 mM sodium azide, pH 7.4. All chemicals
other than polypeptides were purchased from Sigma-Aldrich
(USA).
[0115] ii. Procedures
[0116] Experiments were done using pairs of designed polypeptides,
one negative and one positive. Multilayer films consisting of at
least 5 bi-layers of the above-identified SP2, SNI, LP4, and LN3
were deposited onto the QCM resonators using standard ELBL
techniques (a bi-layer consists of one layer of polycation and one
layer of polyanion). The polypeptide concentration used for layer
adsorption was 2 mg-mL.sup.-1. It is known that dependence of
polyion layer thickness on polyelectrolyte concentration is not
strong (see Lvov, Y. and Decher, G. (1994) Crystallog. Rep. 39:628,
which is incorporated herein by reference in its entirety); in the
concentration range 0.1 to 5 mg mL.sup.-1, bilayer thickness was
approximately independent of concentration for PSS/PAH. By
contrast, polypeptide thin films appear substantially less thick
than those fabricated using high molecular weight PSS/PAH (mass
calculated using .DELTA.f data using the well-known Sauerbrey
equation); see Lvov, Y. and Decher, G. (1994) Crystallog. Rep.
39:628. This follows from calculating film thickness on the basis
of mass deposited, as is ordinarily done in the art for proteins.
The calculated thickness for the designed polypeptide assembly
shown in FIG. 3(c) is greater than the end-to-end length of the
peptides used to make the film. DTT was included at 1 mM to inhibit
disulfide bond formation. The adsorption time was 20 minutes.
[0117] Resonators were rinsed for 1 min. in pure water between
subsequent adsorption cycles (removing perhaps 10-15% of weakly
adsorbed material) and dried in a stream of gaseous N.sub.2. Then
the mass of the deposited peptide was measured indirectly by QCM.
The mass measurement includes some water, despite drying, and low
mass ions like K.sup.+, Na.sup.+, and Cl.sup.-. Partial
interpenetration of neighboring layers of peptide is probable (see
Decher, G. (1997) Science 227:1232; Schmitt et al. (1993)
Macromolecules 26:7058; and Komeev et al. (1995) Physica B
214:954); this could be important for the efficiency of disulfide
"locking."
[0118] iii. Results
[0119] After adsorption of the polypeptide and rinsing and drying
the QCM resonator, the resonant frequency of the resonator was
measured. This enabled calculation of the frequency shift on
adsorption and change in adsorbed mass. A decrease in frequency
indicates an increase in adsorbed mass. The results are provided in
FIGS. 3(a) and 3(b). FIG. 3(a) shows a comparison of adsorption
data for LP4 and LN3 in different buffers (10 mM sodium phosphate,
pH 7.4, 1 mM DTT and 10 mM Tris-HCl, pH 7.4, 1 mM DTT). It is clear
from these data that adsorption depends more on the properties of
the peptides than the specific properties of the buffer used. FIG.
3(b) shows resonator frequency versus adsorbed layer for different
combinations of SP2, SNI, LP4, and LN3 (namely, SP2/SN1, SP2/LN3,
LP4/SN1, and LP4/LN3) in 10 mM sodium phosphate, pH 7.4 and 1 mM
DTT (the lines merely connect experimental data points). Each of
these combinations involved one negative polypeptide and one
positive polypeptide, as required by ELBL. FIG. 3(c) shows a graph
of calculated adsorbed mass versus layer number for SNI and LP4 in
10 mM Tris-HCl, pH 7.4 and 1 mM DTT (calculated from experimental
data using the Sauerbrey equation). The total adsorbed mass,
approximately 5 .mu.g, corresponds approximately to 1 nmol of
peptide. The equation used for this calculation was
.DELTA.m=-0.8710.sup.-9 .DELTA.f, where m is mass in grams and f is
frequency in Hz (see Lvov, Y., Ariga, K., Ichinose, I., and
Kunitake, T. (1995) J. Am. Chem. Soc. 117:6117 and Sauerbrey, G.
(1959) Z. Physik 155:206, both of which are incorporated herein by
reference in their entireties). Film thickness, d, is estimated as
d=-0.016 .DELTA.f where d is in nm (see Yuri Lvov, "Electrostatic
Layer-by-Layer Assembly of Proteins and Polyions" in Protein
Architecture: Interfacial Molecular Assembly and Immobilization
Biotechnology, (Y. Lvov & H. Mohwald eds., 2000) (New York:
Dekker, 2000) pp. 125-167, which is incorporated herein by
reference). The line in FIG. 3(c) is a linear fit to experimental
data points. The linearity of the data is a likely indicator of
precise, regular assembly during adsorption and an approximately
uniform density of the polypeptides in each adsorbed layer.
Adsorption occurred with a frequency shift of -610.+-.60 Hz
(cations) or -380.+-.40 Hz (anions). Linear growth of deposited
polypeptide mass indicates repeatability of adsorption steps early
in the assembly process and the general success of the multilayer
fabrication process.
[0120] iv. Conclusions
[0121] The above results show that polypeptides designed using the
method of the present invention are suitable for ELBL, despite
significant qualitative differences from PSS and PAH, flexible
homopolymers having 1 charge per unit length at pH 7.4. The charge
per unit length on poly-L-lysine and poly-L-glutamic acid is 1 at
pH 7.4, as with PSS and PAH, but both of these polypeptides have a
marked propensity to form .alpha.-helical structure under various
conditions, making them substantially less suitable for multilayer
assembly when control over thin film structure is desired. The
monodisperse polypeptides of the present invention, however, enable
the practitioner to know, quite precisely, the structure of the
material being used for ELBL. Moreover, usual commercial
preparations of poly-L-lysine and poly-L-glutamic acid are
polydisperse, and poly-L-lysine, poly-L-glutamic acid, PSS, and PAH
evoke an immune response (i.e. are immunogenic) in humans.
[0122] Because the designed polypeptides are readily adsorbed on an
oppositely charged surface, as demonstrated by experiment, there is
no need for a "precursor" layer. As is known in the art,
"precursor" layers are deposited on a substrate to enhance
adsorption of less adsorptive substances. The lack of a precursor
layer enhances the biocompatibility of the polyion films because
polymers ordinarily used as precursors are immunogenic or allow
less precise control over polymer structure or thin film structure
than designed polypeptides.
[0123] Multilayers of the designed polypeptides were stable at the
pH of human blood, 7.4. Thus, the multilayers should be useful for
a broad range of biological applications. Adsorption of the
designed polypeptides, each of less than 1 charge per residue, was
essentially complete in less than 10 min. at 2 mg/mL and low ionic
strength. This implies that these polypeptides can be used to form
multilayer films quickly and with relative ease. Drying the peptide
film with N.sub.2(g) after deposition of each layer did not impair
assembly. Drying is done to get an accurate QCM frequency
measurement, but is not required for assembly.
[0124] The film assembly experiments were done at a lower ionic
strength than that of blood, but the process gives a qualitatively
similar result at higher ionic strength. The chief difference is
the amount of peptide deposited per adsorption layer--the higher
the ionic strength, the greater the amount of peptide deposited.
This is illustrated by the graph in FIG. 7, which shows the amount
of material deposited as a function of ionic strength--the peptides
used were poly-L-glutamic acid and poly-L-lysine. QCM resonant
frequency is plotted against adsorption layer. The average
molecular mass of poly-L-glutamate was 84,600 Da, while that of
poly-lys was 84,000 Da. The peptide concentration used for assembly
was 2 mg/mL. The data indicate salt concentration (ionic strength
of solution) influences thin film assembly. In general, the amount
of material deposited per layer increases with ionic strength in
the range 0-100 mM NaCl. As the essential character of ELBL with
designed polypeptides appears not to depend on the choice of buffer
under conditions of relatively high net charge per unit length and
low ionic strength, qualitatively similar results are expected at
the ionic strength of human blood. Thus, the choice of buffer
should not fundamentally alter the stability of the multilayers in
their target environment. However, even if the choice of buffer did
affect the stability of the multilayers, the "locking" mechanism
would be available as a design feature to stabilize the
capsule.
[0125] The greater apparent deposition of positive polypeptides
than negative ones may result from the higher charge per unit
length on the positive polypeptides at pH 7.4. The material
deposited in each layer probably corresponds to that required for
neutralization of the charge of the underlying surface. Hydrophobic
interactions could also help to explain this feature of adsorption
behavior.
[0126] The usual thin film thickness calculation for proteins and
other polymers is probably invalid for short polypeptides
(calculated thickness is 60-90 nm, but summed length of 10
polypeptides is approximately 120 nm). This probably results from a
high density of packing of the relatively short polypeptides onto
the adsorption surface; the result is also consistent with finding
that film thickness varies with ionic strength, as changes in
structural properties of a polymer will occur and screening of
charges by ions will reduce intra-layer charge repulsion between
adsorbed peptides. The thickness of the designed polypeptide thin
film discussed here is estimated at 20-50 nm.
[0127] Many aspects of the design and fabrication cycles could be
automated. For example, a computer algorithm could be used to
optimize the primary structure of peptides for ELBL by comparing
predicted peptide properties with observed physical properties,
including structure in solution, adsorption behavior, and film
stability at extremes of pH. Moreover, the polypeptide film
assembly process can be mechanized, once the details of the various
steps have been sufficiently determined.
2. EXAMPLE 2
Experiments Involving De Novo--Designed Polypeptides Containing
Cysteine
[0128] a. Polypeptides
[0129] The polypeptides used were: TABLE-US-00004 (SEQ ID NO: 5)
Tyr Lys Cys Lys Gly Lys Val Lys Val Lys Cys Lys Gly Lys Val Lys Val
Lys Cys Lys Gly Lys Val Lys Val Lys Cys Lys Gly Lys Val Lys (SEQ ID
NO: 6) Tyr Glu Cys Glu Gly Glu Val Glu Val Glu Cys Glu Gly Glu Val
Glu Val Glu Cys Glu Gly Glu Val Glu Val Glu Cys Glu Gly Glu Val
Glu
Unlike the other polypeptides used in the experiments described
herein, these two were not designed using human genome information;
they were designed for the sole purpose of assessing the role of
disulfide bond formation in polypeptide film stabilization.
[0130] b. Procedures
[0131] All experiments were conducted at ambient temperature.
[0132] All assembly experiments using QCM were conducted in the
same conditions, except that the samples to undergo oxidation were
dried using air instead of nitrogen gas. The assembly conditions
were 10 mM Tris-HCl, 10 mM DTT, pH 7.4. The nominal peptide
concentration was 2 mg/ml. The number of layers formed was 14.
[0133] Disulfide locking conditions for the oxidizing samples were
10 mM Tris-HCl, 1% DMSO, saturation of water with air, pH 7.5. The
duration of the "locking" step was 6 hours. Conditions for the
reducing samples were 10 mM Tris-HCl, 1 mM DTT, saturation of water
with nitrogen, pH 7.5. The duration of this step was 6 hours.
[0134] All disassembly experiments using QCM were conducted in the
same conditions, except that the oxidizing samples were dried using
air instead of nitrogen. Disassembly conditions were 10 mM KCl, pH
2.0 Samples were rinsed with D.I. water for 30 seconds prior to
drying.
[0135] Three different types of experiments were conducted: (1)
Reducing--no treatment: disassembly was conducted immediately after
assembly; (2) Reducing--6 hours, as described above for reducing
samples; and (3) Oxidizing--6 hours, as described above for
oxidizing samples.
[0136] c. Results
[0137] The results are illustrated in FIG. 10. In the first two
experiments (both reducing), all of the deposited material (100%)
disassembled within 50 minutes. By contrast, in the oxidizing
experiment, a substantial amount of material remained after
substantial incubation of the peptide film-coated QCM resonator at
pH 2 for over 5 hours. The stability of the polypeptide films at
acidic pH is determined by the conditions of assembly; in this way,
film or capsule stability is a design feature that becomes possible
by using polypeptides as the polyelectrolytes for ELBL.
[0138] d. Conclusions
[0139] Electrostatic forces play a key role in holding together
oppositely-charged layers of designed polypeptides. At acidic pH,
the net charge on one of the peptides is neutralized and the
polypeptide film disassembles due to electrostatic repulsion.
Reducing conditions prevent disulfide bond formation. Therefore,
the electrostatic attraction between the layers is the only binding
force for stabilizing the layers under these conditions. By
contrast, under oxidizing conditions disulfide bonds are formed. At
acidic pH, disulfide bonds inhibit film disassembly. The results
indicate that layer stability at acidic pH is directly affected by
the formation of intra- and/or inter-layer disulfide bonds--i.e.
between molecules in the same layer, between molecules in adjacent
layers, or both. This is illustrated by the results shown in FIG.
10--due to disulfide locking, more than 30% of the film remained
stable at acidic pH, despite electrostatic repulsion at relatively
low ionic strength. Peptides with more cysteine residues are
anticipated to further improve disulfide locking efficiency.
Moreover, adjustment of the conditions of peptide assembly will be
an important aspect of engineering films to have the desired
physical as well as chemical and biological properties.
3. EXAMPLE 3
Experiments Involving Designed Polypeptides Containing Cysteine
[0140] a. Materials
[0141] The essential elements of this experiment were a quartz
crystal microbalance instrument; silver-coated resonators (9 MHz
resonant frequency); the negative 48-residue peptide (LN3) (SEQ ID
NO: 4); and a positive 48-residue peptide named "SP5" of the
following sequence: TABLE-US-00005 (SEQ ID NO: 7) Tyr Lys Gly Lys
Lys Ser Cys His Gly Lys Gly Lys Lys Ser Cys His Gly Lys Gly Lys Lys
Ser Cys His Gly Lys Gly Lys Lys Ser Cys His
[0142] Like the other designed peptides discussed above in Part
VII(E)(1), SP5 was designed using the process described above in
Part VII(B)(1) to analyze the amino acid sequence of the human
blood protein lactotransferrin (gi|4505043). The ELBL buffer was 10
mM Tris, pH 7.4, 10 mM NaCl, and 1 mM DTT. The disassembly buffer
was 10 mM KCl, pH 2. 2 mL peptide solutions were prepared for SP5
and LN3 by adding 4 mg of each peptide to 2 mL of the above buffer
solution and adjusting the pH of each solution to 7.4; the peptide
concentration was 2 mg-mL.sup.-
[0143] b. Procedure for Monitoring Assembly of Polypeptide Layers
on QCM Resonators
[0144] Reducing procedures were as follows: (1) The frequency of
the resonator was measured and recorded prior to peptide
adsorption; (2) The resonator was dipped into the SP5 peptide
solution for 20 min.; (3) The resonator was dipped into the SP5
rinse solution for 30 sec.; (4) The resonator was removed from the
rinse solution and dried using nitrogen gas; (5) The QCM resonant
frequency of the resonator was recorded; (6) The resonator was
dipped into the LN3 peptide solution for 20 min.; (7) The resonator
was dipped into the LN3 rinse solution for 30 sec.; (8) The
resonator 1 was removed from the rinse solution and dried using
nitrogen gas; (9) The QCM resonant frequency of the resonator was
recorded; (10) Steps 2 through 9 were repeated until 16 layers were
adsorbed onto the resonator.
[0145] Oxidizing procedures were the same as the reducing
procedures, except that the resonator was rinsed in D.I. water
instead of the SP5 buffer or the LN3 buffer and dried with air
instead of nitrogen before each measurement.
[0146] c. Locking Procedures
[0147] Reducing procedures were as follows: The resonator was
placed in an aqueous solution containing 1 mM DTT for 6 hours. DTT,
a reducing agent, inhibited disulfide bond formation.
[0148] Oxidizing procedures were as follows: The resonator was
placed in an air-saturated aqueous solution containing 1% DMSO for
6 hours. DMSO, an oxidizing agent, promoted disulfide bond
formation.
[0149] d. Disassembly on Resonator
[0150] i. Solutions
[0151] Reducing conditions were as follows: 10 mM KCl, 1 mM DTT, pH
2.
[0152] Oxidizing conditions were as follows: 10 mM KCl, 20% DMSO,
pH 2.
[0153] ii. Procedure for Disassembly
[0154] Reducing procedures were as follows: (1) The initial
resonant frequency of the resonator was measured by QCM and
recorded; (2) The resonator was dipped into the reducing
disassembly solution for 5 min.; (3) The resonator was rinsed in
reducing buffer solution for 30 sec.; (4) The resonator was dried
with gaseous N.sub.2; (5) The resonant frequency of the resonator
was measured by QCM and recorded; (6) Steps 2 through 5 were
repeated for reading times of 5, 10, 15, 20, 30, 60, and 90
min.
[0155] Oxidizing procedures were the same as for reducing
procedures, except that rinsing of the resonator was done in D.I.
water saturated with air instead of reducing buffer.
[0156] e. Results
[0157] FIG. 8 shows approximately linear increase in mass deposited
during thin film assembly of SP5 and LN3. Both resonators show
almost identical deposition of mass throughout the process of
assembly, despite differences in assembly conditions.
[0158] FIG. 9 shows the percentage of material remaining during
film disassembly. The layers subjected to oxidizing conditions
showed a minimal loss of material at acidic pH with almost 90 to
95% of mass retention. By contrast, layers subjected to reducing
conditions lost almost all the film material within the first 5
minutes of exposure to acidic pH.
[0159] f. Conclusions
[0160] The results demonstrate that at acidic pH, disulfide bonds
prevent layer degeneration and hold the layers firmly together.
Layer stability at acidic pH is directly affected by the formation
of intra- and/or inter-layer disulfide bonds. Disulfide bond
formation is dependent on the concentration and proximity of
cysteine residues to each other. Increasing the concentration per
unit chain length of the polypeptide would therefore directly
influence disulfide bond formation and thin film stability.
Increasing the ionic strength of the buffer solutions used for film
assembly influences the concentration of cysteine in the film by
increasing the amount of material deposited per adsorption cycle
and the thickness of each layer. The increased number of cysteine
amino acids in a single layer would in this way increase the number
of disulfide bonds formed, and, on oxidation, increase film
stability.
[0161] Other embodiments of the invention are possible and
modifications may be made without departing from the spirit and
scope of the invention. Therefore, the detailed description above
is not meant to limit the invention. Rather, the scope of the
invention is defined by the appended claims.
Sequence CWU 1
1
8 1 32 PRT Homo sapiens 1 Tyr Arg Arg Arg Arg Ser Val Gln Gly Arg
Arg Arg Arg Ser Val Gln 1 5 10 15 Gly Arg Arg Arg Arg Ser Val Gln
Gly Arg Arg Arg Arg Ser Val Gln 20 25 30 2 32 PRT Homo sapiens 2
Tyr Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln Asp 1 5
10 15 Gly Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp Glu Cys Gln
Asp 20 25 30 3 48 PRT Homo sapiens 3 Tyr Arg Arg Arg Arg Ser Val
Gln Gly Arg Arg Arg Arg Ser Val Gln 1 5 10 15 Gly Arg Arg Arg Arg
Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln 20 25 30 Gly Arg Arg
Arg Arg Ser Val Gln Gly Arg Arg Arg Arg Ser Val Gln 35 40 45 4 48
PRT Homo sapiens 4 Tyr Glu Glu Asp Glu Cys Gln Asp Gly Glu Glu Asp
Glu Cys Gln Asp 1 5 10 15 Gly Glu Glu Asp Glu Cys Gln Asp Gly Glu
Glu Asp Glu Cys Gln Asp 20 25 30 Gly Glu Glu Asp Glu Cys Gln Asp
Gly Glu Glu Asp Glu Cys Gln Asp 35 40 45 5 32 PRT Homo sapiens 5
Tyr Lys Cys Lys Gly Lys Val Lys Val Lys Cys Lys Gly Lys Val Lys 1 5
10 15 Val Lys Cys Lys Gly Lys Val Lys Val Lys Cys Lys Gly Lys Val
Lys 20 25 30 6 32 PRT Homo sapiens 6 Tyr Glu Cys Glu Gly Glu Val
Glu Val Glu Cys Glu Gly Glu Val Glu 1 5 10 15 Val Glu Cys Glu Gly
Glu Val Glu Val Glu Cys Glu Gly Glu Val Glu 20 25 30 7 32 PRT Homo
sapiens 7 Tyr Lys Gly Lys Lys Ser Cys His Gly Lys Gly Lys Lys Ser
Cys His 1 5 10 15 Gly Lys Gly Lys Lys Ser Cys His Gly Lys Gly Lys
Lys Ser Cys His 20 25 30 8 23 PRT Homo sapiens 8 Asp Ser His Ala
Lys Arg His His Gly Tyr Lys Arg Lys His Glu Lys 1 5 10 15 His His
Ser His Arg Gly Tyr 20
* * * * *
References