Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Scleroproteins – e.g. – fibroin – elastin – silk – etc.
Reexamination Certificate
2001-06-28
2003-11-04
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Scleroproteins, e.g., fibroin, elastin, silk, etc.
C530S300000
Reexamination Certificate
active
06642361
ABSTRACT:
The sequence listing information submitted on computer readable form is identical to the written sequence listing contemporaneously submitted on paper, and includes no new matter.
FIELD OF THE INVENTION
The present invention relates to the cocoon silk protein isolated from the black fly,
Simulium vittatum.
BACKGROUND OF THE INVENTION
Silk is a natural, protein filament fiber. Several types of natural silk that are known to date are excreted by invertebrates such as those that belong to two classes of the phylum Arthropoda: Insecta and Arachnida. Silk producing insects include silk worms, black flies, wasps, and lacewing flies.
Some arthropods' silk have now been cloned. For example, Lewis, R. V. et al. (U.S. Pat. No. 5,728,810) teach the preparation of spider silk protein by recombinant DNA techniques. Lewis, R. V., et al. (U.S. Pat. No. 5,733,771) teach a cDNA encoding minor ampulate silk proteins. Lombardi, S. J. et al. (U.S. Pat. No. 5,245,012) teach a recombinant spider silk protein which can be obtained in a commercially useful form by the cloning of host cells encoding such protein.
Another silk producing arthropod, the black fly, evolved to produce a very durable silk filament. Silk is produced by the black fly “larva” which forms a cocoon. The larva and pupae are aquatic but are confined to running waters where they attach themselves to firm substrates. The black fly's silk filament is able to withstand the exposure to water flow in order to keep the pupa inside the cocoon intact. Another remarkable property of the Simuliidae silk is its ability to maintain its adhesive characteristic while submerged in water. These properties are very attractive in terms of possible application of the black fly silk as a biomaterial.
The above prior art references are incorporated herein by reference.
The prior art does not teach the isolation of a nucleic acid molecule coding for the silk protein from black flies. Further, the prior art does not teach the expression of such silk protein using recombinant DNA techniques.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides an isolated polypeptide molecule having an amino acid sequence of SEQ ID NO: 1. In a further embodiment, the present invention provides an isolated nucleic acid molecule coding for such polypeptide. In another embodiment, the present invention provides the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 6. In a further embodiment, the present invention provides a polypeptide molecule having an amino acid sequence of SEQ ID NO: 7 and expressed by such nucleic acid molecule. In yet another embodiment, the present invention provides an isolated nucleic acid molecule coding for such polypeptide. In addition, the present invention provides a cloning vector comprising the nucleotide sequence of SEQ ID NO: 6 and a host cell transformed with such vector. In a further embodiment, the present invention provides a fiber formed from polypeptide of SEQ ID NO: 7. In yet another embodiment, the present invention provides a method of isolating cocoon silk protein comprising the steps of: a) boiling a cocoon in a sample reducing buffer to remove SDS (sodium dodecyl sulfate)-soluble proteins, b) centrifuging the sample, c) withdrawing supernatant, adding formic acid to the pellet and incubating the sample in order to solubilize SDS-insoluble proteins, d) freezing and lyophilizing the sample in order to freeze-dry the sample, e) re-suspending the dried sample in a buffer to protect proteins against potential residual proteolytic activity for subsequent analysis using SDS-PAGE (polyacrylamide gel electrophoresis).
REFERENCES:
patent: 5245012 (1993-09-01), Lombardi et al.
patent: 5728810 (1998-03-01), Lewis et al.
patent: 5733771 (1998-03-01), Lewis et al.
patent: WO 97/08315 (1997-03-01), None
Tokutake, S. (1980) Isolation of the smallest component of silk protein. Biochem. J. vol. 187, pp. 413-417.
Bidochka Michael J.
Hunter Fiona F.
Carlson Karen Cochrane
Goodwin & Procter LLP
Liu Samuel Wei
LandOfFree
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