Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1995-06-07
2001-02-06
Yucel, Remy (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S353000, C435S069100
Reexamination Certificate
active
06184348
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention is related to the production of high molecular weight polymers of amino acids based on biologically and chemically active structural polymers.
2. Background
Recombinant DNA technology has been applied in the isolation of natural genes and the expression of these genes in a variety of host cells. Typically, this technology has had utility in producing biologically active polypeptides, such as interferons or peptide hormones, which were impractical to produce in useful amounts by other means. It was also possible to produce modified proteins by isolating natural genes and utilizing the techniques of site specific, in vitro. mutagenesis to alter these genes and thereby change the polypeptides produced. Other polypeptides have been created by combining sections of various native genes to produce new polypeptides that are chimeric molecules of the several naturally occurring molecules.
With the advent of efficient and automated methods for the chemical synthesis of DNA, it has become possible to synthesize entire genes and to modify such synthetic genes at will during the course of synthesis. However, these various technologies have been applied to the production of natural or modified versions of natural polypeptides. There have been very few attempts to use these technologies to create substantially new polypeptides. In nature, polypeptides have a wide range of chemical, physical and physiological characteristics. Nevertheless there are commercial applications for which known, naturally occurring polypeptides are not appropriate.
While biotechnology is versatile, usually it has been limited in its applications to naturally occurring products or modifications of naturally occurring molecules. One great strength of organic chemical synthesis, by contrast, has been the ability to transform inexpensive carbon materials to a wide variety of polymeric molecules, including naturally occurring molecules, but most importantly entirely new chemical structures, such as polypropylene and polyacrylates, which have defined and predicted chemical properties not associated with naturally occurring molecules.
Such materials, particularly high-molecular weight polymers containing repeating sequences of amino acids, have proven difficult to produce by biochemical means. The genes necessary for producing large peptides containing repeating units of amino acids were unstable and often underwent intermolecular recombination causing deletions of repeating units in the gene. The development of a biotechnology which should produce polymeric molecules by biological processes similar to those available by organic synthesis would significantly broaden the range of applications of biotechnology.
BRIEF DESCRIPTION OF THE RELEVANT LITERATURE
The cloning of multiple lactose operators up to four in tandem is disclosed by Sadler et al.,
Gene,
(1980) 8:279-300. Hybrid bacterial plasmids containing highly repeated satellite DNA is disclosed by Brutlag et al.,
Cell,
(1977) 10:509-519. The synthesis of a poly(aspartyl-phenylalanine) in bacteria is disclosed by Doel et al.
Nucleic Acids Research,
(1980) 8:4575-4592. A method for enriching for proline content by cloning a plasmid which codes for the production of a proline polymer was disclosed by Kangas et al.,
Applied and Environmental Microbiology,
(1982) 43:629-635. The biological limitations on the length of highly repetitive DNA sequences that may be stably maintained within plasmid replicons is discussed by Gupta et al. in
Bio/Technology,
p. 602-609, September 1983.
SUMMARY OF THE INVENTION
Methods and compositions are provided for the production of polypeptides having repetitive oligomeric units or strands which interact forming a structural component, where the structural components are separated by different amino-acid sequences having particular functional capabilities. (By “strands” is intended an ordered sequence capable of alignment with a second strand having substantially the same or a complementary sequence e.g., hydrophobic aligns with hydrophobic and hydrophilic aligns with hydrophilic.) The components of the repetitive units provide a structure which allows for availability of the intervening units for interacting with the environment, such as solutions, gases, gels and the like where the intervening sequence is substantially free of steric inhibition to interact with other molecules as compared to the strands. Long nucleic-acid sequences are built up by synthesizing nucleic-acid oligomers which express a plurality of individual repetitive peptide units and the oligomers are joined to provide a polynucleotide of the desired length. By providing for specific restriction sites which are relatively evenly spaced, additional sequences may be introduced at sites which provide for turns or other functional entity between repetitive strands. The resulting nucleic-acid open-reading frames may then be introduced into an appropriate expression vector for expression of the desired protein product.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel polypeptides are provided which are polyoligomers of repeating, relatively short, amino acid sequence units forming strands, where the strands are separated by a different oligomeric unit which results in a sequence available to the environment of the polypeptide for a variety of functions.
The polypeptides which are encoded for by the nucleic acid sequences of the subject invention provide for strands which may align to provide for a basic structure. The strands will be protein chain segments of more or less linear conformation. The strands may be aligned in reverse or direct alignment, where a plurality of strands are separated by a sequence other than the repetitive sequence as a functional entity between the strands, so as to be accessible to solutes or components in the medium. The strands will be based primarily on repeating units of naturally occurring polymers, where the repeating units generally are at least three amino acids and may include the same amino acid twice.
The polymers of this invention may be used to provide a variety of structures for a variety, of purposes. Depending on the repeating units and their relationship to known polymeric structures, analogous mechanical, e.g., tensile properties, may be achieved, which may be modified for a particular intended purpose. The subject polymers may be used by themselves to produce various articles, including formed objects, coatings, or other structural or non-structural components including fibers, films, membranes, adhesives, emulsions, and the like or with other compounds or compositions to form composites, laminates or combinations of the aforementioned products.
The structurally aligned elements of the polymers may be &bgr;-sheets, &agr;-helices, dynamic &bgr;-spirals, collagen helices, Type I or II, or combinations thereof, where there will be intervening elements which will be of different sequence and structure and will usually not participate in the aligned elements. For the most part, these intervening sequences will be loops or turns.
The genes of the subject invention comprise multimers of DNA sequences encoding the same amino acid sequence unit, where two or more different multimers encoding different amino acid units may be joined together to form a block copolymer. The individual units will have from 3 to 30 amino acids (9 to 90 nt), more usually 3 or 4 to 25 amino acids (9 to 75 nt), particularly 3 or 4 to 15 amino acids (9 to 45 nt), more particularly 3 or 4 to 9 amino acids (9 to 27 nt), usually having the same amino acid appearing at least twice in the same unit, generally separated by at least one amino acid. The units of the multimer coding for the same amino acid sequence may involve two or more nucleotide sequences, relying on the codon redundancy to achieve the same amino acid sequence.
The units of the strands will generally be at least about 25 amino acids and not more than about 200 amino acids, usually not more than about 100 amino acids, preferably from about 30 to 75 amino
Cappello Joseph
Ferrari Franco A.
Flehr Hobach Test Albritton & Herbert LLP
McGarry Sean
Protein Polymer Technologies
Trecartin Richard F.
Yucel Remy
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