Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1999-03-19
2003-01-14
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S028000, C435S195000, C525S054100, C525S054110, C106S124100, C106S124300, C106S160100, C530S211000, C530S338000, C530S343000, C530S402000, C530S407000
Reexamination Certificate
active
06506577
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to adhesive copolymers modeled on bioadhesive proteins secreted by marine organisms. These copolymers are compatible with the metabolism, growth and function of living tissues and/or cells in vitro or in vivo and, consequently, are suitable for use in a wide variety of biomedical applications.
2. Description of Related Art
Polymeric materials have been widely used for implants or other biomedical applications, since they bear close resemblance to natural tissue components such as collagen, which allows direct bonding with other substances. Decades of peptide research have created a wide variety of biomedically useful polypeptides. However, they still are the most underrated and underused polymers considering their impressive properties, which include infusibility, mechanical strength and adhesive capability due to a highly flexible backbone and many functional side chains. Waite, et al.,
Science
, 212:1038-1040 (1981) identified some of nature's most powerful adhesives, bioadhesive polyphenolic proteins, secreted by marine mussels which live under water and routinely cope with the forces of surf and tides. The naturally-occurring bioadhesive polyphenolic protein is produced and stored in the exocrine phenol gland of the mussel and is deposited onto marine surfaces by the mussel's foot during the formation of new adhesive plaques. While the natural bioadhesive polyphenolic protein can be extracted and purified according to the procedures set forth in the
Journal of Biological Chemistry
, 258:2911-2915 (1983) or U.S. Pat. No. 4,496,397, the utility of the natural bioadhesive polyphenolic protein extracted from the mussel is limited by the quantities that can be obtained. Consequently synthetic polymers of these natural bioadhesive proteins have been the focus of a significant amount of research, particularly for their potential use as surgical tissue adhesives.
Decades of investigation into this field has led to the discovery of many different marine organisms which secrete adhesive materials. These organisms include many varieties of mussels, which have different environmental needs and subsequent uses for the adhesives they produce, but are alike in that the materials they use for adhesion and cementing contain many of the same building blocks and apparently operate by the same mechanism. See e.g. J. H. Waite, et al.,
J. Comp. Physiol. B,
159:517-525 (1989) and L. M. Rzepecki, et al.,
Mol. Mar. Biol. Biotech.,
2:255-279 (1993).
The adhesive proteins isolated from mussels (e.g., Mytilus edulis) have been purified and examined for use as tissue adhesives. Preliminary experiments indicated that these proteins are very effective for formation of adhesive bonds to animal tissues and also exhibit low immunogenicity. See e.g. J. H. Waite,
Polym. Prepr.,
30(1):181-182 (1990) and C. Saez, et al.,
Comp. Biochem. Physiol.,
98B:569-572 (1991). The major drawbacks with these materials are that (i) their mechanisms of action are poorly understood, (ii) the essential requirements for good adhesion and crosslinking are unknown, (iii) recombinant proteins must be enzymatically treated to generate post-translationally modified residues (i.e. DOPA), and (iv) these proteins cannot be produced inexpensively or in the large quantities necessary for successful commercial application. See e.g. J. H. Waite,
Biol. Rev.,
58:209-231 (1983) and C. V. Benedict, et al.,
ACS Symp. Ser.,
385:465-483 (1989).
The adhesive precursor proteins have been isolated and sequenced from a wide variety of organisms and are known to show certain characteristics. A partial list of these proteins is given in FIG.
1
. Examples of amino acid structures found in adhesive proteins are provided in FIG.
2
. It is important to note that these consensus repeats are just that, and that considerable variation is present in the sequence of each protein. The repetitive polypeptides have basic isoelectric points (due to lysine residues), flexible conformations (due to high percentages of small glycine and serine residues), and high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA). See e.g. J. H. Waite, et al.,
Science,
212:1038-1040 (1981) and J. H. Waite,
J. Biol. Chem.,
258:2911-2915 (1983). The DOPA residues are believed to be primarily responsible for (i) chemisorption of the polymers to surfaces underwater and (ii) covalent cross-linking or setting of the adhesive. J. H. Waite,
Comp. Biochem. Physiol.,
97B:19 (1990); J. H. Waite,
Biol. Bull.,
183:178-184 (1992).
A number of groups have studied the bioadhesive qualities of synthetic peptides modeled upon marine adhesive proteins. Yamamoto, et al. report the synthesis of L-DOPA homopolymer as well as copolymers of L-DOPA with L-lysine and L-glutamic acid. H. Yamamoto, et al.,
Polymer,
19:1115-1117 (1978); H. Yamamoto, et al.,
Macromolecules,
16:1058-1063 (1983); H. Yamamoto, et al.,
Biopolymers,
21:1137-1151 (1982); H. Yamamoto, et al.
Biopolymers,
18:3067-3076 (1979). They have also reported random L-lysine/L-tyrosine copolymers, and the synthesis of complex random copolypeptides which contain as many as 18 different amino acids, including L-DOPA. H. Yamamoto, et al.,
Int. J. Biol. Macromol.,
12:305-310 (1990); A. Nagai, et al.,
Bull. Chem. Soc. Japn.,
62:2410-2412 (1989); H. Yamamoto, et al.,
Mar. Chem.,
37:131-143 (1992); H. Yamamoto, et al.,
Mar. Chem.,
26:331-338 (1989). Sequentially specific copolymers between L-DOPA and L-lysine or L-glutamic acid were prepared by stepwise condensation procedures. Random copolymers of L-DOPA and L-glutamic acid were prepared by polymerization of N-carboxyanhydride (NCA) monomers. Benedict et. al. at Biopolymers, Inc. have also reported synthetic polymers which were composed of small L-DOPA containing peptides grafted to polyamine backbones. C. B. Benedict, et al., U.S. Pat. No. 4,908,404, Mar. 13, 1990. The materials were reported to form adhesive bonds to a variety of substrates and were found to work well with phosphate buffers.
Studies on a variety of synthetic polypeptides modeled upon marine adhesive proteins indicate that these molecules are promising candidates for use as bioadhesives. H. Yamamoto,
J. Chem. Soc. Perkin Trans. I,
613-918 (1987). Additional studies disclose an analysis of adhesive properties of L-lysine/L-tyrosine random copolymers and complex random copolymers where tyrosinase enzyme was used as an oxidizing agent. H. Yamamoto, et al.,
Int. J. Biol. Macromol.,
12:305-310 (1990); A. Nagai, et al.,
Bull. Chem. Soc. Japn.,
62:2410-2412 (1989). These adhesive systems were, however, studied under limited reaction environments such as water and diluted synthetic seawater and were found to form adhesive bonds to iron and Al
2
O
3
.
In order to facilitate the commercial applications of synthetic peptides modeled upon marine adhesive proteins, there is a need for methods to precisely control the material aspects of the adhesive matrix. In particular, such controlled manipulation of adhesive polypeptide characteristics such as curing time and adhesive strength have a wide number of applications in different biomedical and related commercial contexts. Unfortunately, while the manipulation of adhesive polypeptide characteristics have a number of biomedical applications, specific methods of controlling the material properties of these molecules have not been disclosed.
SUMMARY OF THE INVENTION
To address the need for methods to control the crosslinking of synthetic peptides modeled upon marine adhesive proteins, we disclose methods which allow us to synthesize and manipulate the characteristics of copolypeptides which contain the side-chain functional groups (e.g. catechol and primary amine) that are present in these natural adhesive proteins. Specifically, we examined a range of different copolymers to illustrate the effects of a number of factors on crosslinking behavior including the oxidizing agent, copolypeptide concentration and composition and sequence of the functional group
Deming Timothy J.
Yu Miaoer
Fulbright & Jaworski L.L.P.
Low Christopher S. F.
Mohamed Abdel A.
The Regents of the University of California
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