Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-12-11
2001-07-31
Saoud, Christine J. (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S252310, C435S252330, C435S252350, C435S254200, C435S254230, C435S254300, C435S320100, C530S300000, C530S353000, C536S023100, C536S023400
Reexamination Certificate
active
06268169
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel spider silk protein analogs derived from the amino acid consensus sequence of repeating units found in the natural spider dragline of
Nephila clavipes
. More specifically, synthetic spider dragline has been produced from
E. coli
and
Bacillus subtilis
recombinant expression systems wherein expression from
E. coli
is at levels greater than 1 mg full-length polypeptide per gram of cell mass.
BACKGROUND
Ever increasing demands for materials and fabrics that are both light-weight and flexible without compromising strength and durability has created a need for new fibers possessing higher tolerances for such properties as elasticity, denier, tensile strength and modulus. The search for a better fiber has led to the investigation of fibers produced in nature, some of which possess remarkable qualities. The virtues of natural silk produced by
Bombyx mori
(silk worm) have been well known for years but it is only recently that other other naturally produced silks have been examined.
Spider silks have been demonstrated to have several desirable characteristics. The orb-web-spinning spiders can produce silk from six different types of glands. Each of the six fibers has different mechanical properties. However, they all have several features in common. They are (i) composed predominantly or completely of protein; (ii) undergo a transition from a soluble to an insoluble form that is virtually irreversible; (iii) composed of amino acids dominated by alanine, serine, and glycine and have substantial quantities of other amino acids, such as glutamine, tyrosine, leucine, and valine. The spider dragline silk fiber has been proposed to consist of pseudocrystaline regions of antiparallel, &bgr;-sheet structure interspersed with elastic amorphous segments.
The spider silks range from those displaying a tensile strength greater than steel (7.8 vs 3.4 G/denier) and those with an elasticity greater than wool, to others characterized by energy-to-break limits that are greater than KEVLAR® (1×10
5
vs 3×10
4
JKG-1). Given these characteristics spider silk could be used as a light-weight, high strength fiber for various textile applications.
Considerable difficulty has been encountered in attempting to solubilize and purify natural spider silk while retaining the molecular-weight integrity of the fiber. The silk fibers are insoluble except in very harsh agents such as LiSCN, LiClO4, or 88% (vol/vol) formic acid. Once dissolved, the protein precipitates if dialyzed or if diluted with typical buffers. Another disadvantage of spider silk protein is that only small amounts are available from cultivated spiders, making commercially useful quantities of silk protein unattainable at a reasonable cost. Additionally, multiple forms of spider silks are produced simultaneously by any given spider. The resulting mixture has less application than a single isolated silk because the different spider-silk proteins have different properties and, due to solubilization problems, are not easily separated by methods based on their physical characteristics. Hence the prospect of producing commercial quantities of spider silk from natural sources is not a practical one and there remains a need for an alternate mode of production. The technology of recombinant genetics provides one such mode.
By the use of recombinant DNA technology it is now possible to transfer DNA between different organisms for the purposes of expressing desired proteins in commercially useful quantities. Such transfer usually involves joining appropriate fragments of DNA to a vector molecule, which is then introduced into a recipient organism by transformation. Transformants are selected by a known marker on the vector, or by a genetic or biochemical screen to identify the cloned fragment. Vectors contain sequences that enable autonomous replication within the host cell, or allow integration into a chromosome in the host.
If the cloned DNA sequence encodes a protein, a series of events must occur to obtain synthesis of this foreign protein in an active form in the host cell. Promoter sequences must be present to allow transcription of the gene by RNA polymerase, and a ribosome binding site and initiation codon must be present in the transcribed mRNA for translation by ribosomes. These transcriptional and translational recognition sequences are usually optimized for effective binding by the host RNA polymerase and ribosomes, and by the judicious choice of vectors, it is often possible to obtain effective expression of many foreign genes in a host cell.
While many of the problems of efficient transcription and translation have been generally recognized and for the most part, overcome, the synthesis of fiber-forming foreign polypeptides containing high numbers of repeating units poses unique problems. Genes encoding proteins of this type are prone to genetic instability due to the repeating nucleic acid sequences. Ideally, they encode proteins of high molecular weight, consisting of at least 800 amino acid residues, and generally with restricted amino acid compositions. While
E. coli
produces endogenous proteins in excess of 1000 residues, production of long proteins of restricted amino acid composition appears to place an unbalanced strain on the biosynthetic system, resulting in the production of truncated products, probably due to abortive translation.
In spite of the above mentioned difficulties, recombinant expression of fiber forming proteins is known in the art. Chatellard et al.,
Gene
, 81, 267, (1989) teach the cloning and expression of the trimeric fiber protein of human adenovirus type 2 from
E. coli
. The gene expression system relied upon bacteriophage T7 RNA polymerase and optimal gene expression was obtained at 30° C. where the foreign protein attained levels of 1% of total host protein.
Goldberg et al.,
Gene
, 80, 305, (1989) disclose the cloning and expression in
E. coli
of a synthetic gene encoding a collagen analog (poly (Gly-Pro-Pro)). The largest DNA insert was on the order of 450 base pairs and it was suggested that large segments of highly-repeated DNA may be unstable in
E. coli.
Ferrari et al. (WO 8803533) disclose methods and compositions for the production of polypeptides having repetitive oligomeric units such as those found in silk-like proteins and elastin-like proteins by the expression of synthetic structural genes. The DNA sequences of Ferrari encode peptides containing an oligopeptide repeating unit which contains at least 3 different amino acids and a total of 4-30 amino acids, there being at least 2 repeating units in the peptide and at least 2 identical amino acids in each repeating unit.
Cappello et al. (WO 9005177) teach the production of a proteinaceous polymer from transformed prokaryotic hosts comprising strands of repeating units which can be assembled into aligned strands and DNA sequences encoding the same. The repeating units are derived from natural polymers such as fibroin, elastin, keratin or collagen.
The cloning and expression of silk-like proteins is also known. Ohshima et al.,
Proc. Natl. Acad. Sci. U.S.A
., 74, 5363, (1977) reported the cloning of the silk fibroin gene complete with flanking sequences of
Bombyx mori
into
E. coli
. Petty-Saphon et al. (EP 230702) disclose the recombinant production of silk fibroin and silk sericin from a variety of hosts including
E. coli, Saccharomyces cerevisiae
, Pseudomonas sp Rhodopseudomonas sp, Bacilus sp, and Streptomyces sp. In the preferred embodiments the expression of silk proteins derived from
Bombyx mori
is discussed.
Progress has also been made in the the cloning and expression of spider silk proteins. Xu et al.,
Proc. Natl, Acad. Sci. U.S.A
., 87, 7120, (1990) report the determination of the sequence for a portion of the repetitive sequence of a dragline silk protein, Spidroin 1, from the spider
Nephila clavipes
, based on a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of two dif
E. I. Du Pont de Nemours and Company
Saoud Christine J.
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