Spider dragline silk is one of most perfect fibrous proteins in nature. As biomaterials, it has a wide application in tissue engineering due to its unique mechanical properties, good biocompatibility, slow degradation. In this paper, based on the highly repetitive sequence of spider dragline silk and with the introduced RGD peptide codons which involve cell adhesion, the DNA monomer sequence encoding RGD-spider dragline silk was synthesized, and then was used to construct the multimers by the strategy of "head to tail"; the multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLyS, were transformed the expression of recombinant protein was induced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 35KD and 60KD respectively in agreement with the desired. Western assay was used for determining the specification of products. Further, the purification process was groped for the producing of large quantity of synthetic proteins through high density fermentation.
Amino Acid Sequence ; Animals ; Base Sequence ; Biocompatible Materials ; Cloning, Molecular ; Fibroins ; biosynthesis ; genetics ; isolation & purification ; Molecular Sequence Data ; Oligopeptides ; biosynthesis ; isolation & purification ; Polymers ; isolation & purification ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Spiders

Spider silk becomes the protein fibroin with excellent character in the natural world because of its unique mechanical properties. The genetic engineering technique is an effective method to procure the spider silk protein. In order to obtain RGD-spider silk protein gene recombinant engineering strain pNSR-16 on a regular scale, we determine the optimal conditions of the growth and expression of pNSR-16 through culture in laboratory, and based on these, study the fed-batch high density culture. We control the flow of carbon-nitrogen, the dissolved oxygen concentration and the cell specific growth rate, which make the final cell density and concentration of recombinant protein of total protein arrive at OD600 57.15 and 20.8%, respectively.
Animals ; Bacteria ; genetics ; Cloning, Molecular ; Fermentation ; Fibroins ; biosynthesis ; genetics ; Genetic Engineering ; Oligopeptides ; genetics ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; genetics