Production of silk-like proteins in plants

#The invention relates to the field of molecular biology and plant genetics. More specifically, this invention describes a technique to produce silk-like proteins plant expression systems.

#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. One of those fibers is silk, a group of externally spun fibrous protein secretions.

#Silks are produced by over 30,000 species of spiders and by many other insects particularly in the order Lepidoptera (Foelix, R. F. (1992) Biology of Spiders, Cambridge, Mass. Harvard University Press). Few of these silks have been studied in detail. The cocoon silk of the domesticated silkworm Bombyx mori and the dragline silk of the orb-weaving spider Nephila clavipes are among the best characterized. Although the structural proteins from the cocoon silk and the dragline silk are quite different from each other in their primary amino acid sequences, they share remarkable similarities in many aspects. They are extremely glycine and alanine-rich proteins. Fibroin, a structural protein of the cocoon silk, contains 42.9% glycine and 30% alanine. Spidroin 1, a major component of the dragline silk, contains 37.1% glycine and 21.1% alanine. They are also highly repetitive proteins. The conserved crystalline domains in the heavy chain of the Fibroin and a stretch of polyalanine in Spidroin 1, are repeated numerous times throughout entire molecules. These crystalline domains are surrounded by larger non-repetitive amorphous domains in every 1 to 2 kilobases in the heavy chain of Fibroin, and by shorter repeated GXG amorphous domains in tandem in Spidroin 1. They are also shear sensitive due to their high copy number of the crystalline domains. During fiber spinning, the crystalline repeats are able to form anti-parallel β-pleated sheets, so that silk protein is turned into semi-crystalline fiber with amorphous flexible chains reinforced by strong and stiff crystals (Kaplan et al., (1997) in Protein-Based Materials, McGrath, K., and Kaplan, D. Eds, Birkhauser, Boston, pp 104-131).

#FIG. 1 is a plasmid map of pGY001 carrying the GYS adapter.

#FIG. 2A is a plasmid map of pGY101 carrying the DP-1B.8P gene.

#FIG. 2B is a plasmid map of pGY102 carrying the DP-1B.16P gene.

#Applicants made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure: 1 Depositor Identification International Date of Reference Depository Designation Deposit pGY401 ATCC PTA-1912 May 24, 2000 pLS3 ATCC PTA-1911 May 24, 2000

#Applicant(s) have provided 29 sequences in conformity with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. 2 SEQ ID NO: SEQ ID NO: Sequence Description Nucleic acid Amino acid Spirdroin 1 1 SLP repeat unit 2 SLP repeat unit 3 Peptide SLP 4 SLP repeat unit 5 SLP repeat unit 6 SLP repeat unit 7 Spider silk variant 8 Spider silk variant repeat unit 9 DP-1A 10 DP-1B 11 Spider silk repeat unit 12 DP-1B 809 amino acid repeat 13 DP-1B 1617 amino acid repeat 14 Primer 15 Primer 16 Peptide adapter 17 Sense DNA strand encoding 18 a peptide adapter Anti-sense DNA strand encoding 19 a peptide adapter Adapter peptide 20 Gene encoding DP-1B 8-mer 21 DP-1B 8mer 22 Gene encoding DP-1B 16-mer 23 DP-1B 16-mer 24 Spider silk repeat unit 25 Primer 26 Primer 27 Primer 28 Primer 29

#The present invention provides methods for of the production of silks and silk-like proteins in green plants. The methods allow for the more cost effective production of silk heretofore not obtainable from natural or microbial sources. The silks and silk-like proteins of the present invention may have properties suitable for fabrics, or alternatively may be useful in materials construction. For example the spider dragline silk has a tensile strength of over 200 ksi with an elasticity of nearly 35%, which makes it more difficult to break than either KEVLAR® fibers or steel. When spun into fibers, spider silk may have application in the bulk clothing industries as well as being applicable for certain kinds of high strength uses such as rope, surgical sutures, flexible tie downs for certain electrical components and even as a biomaterial for implantation (e.g., artificial ligaments or aortic banding). Additionally these fibers may be mixed with various plastics and/or resins to prepare a fiber-reinforced plastic and/or resin product.