Artificial Synthesis Spider Silk Protein MaSP2

Spider silk has a high performance and attracting many people to study on it. Beacause of the spiders are strong, aggressive and cannibalistic, it is hard to feed them like silkworm to product silk by itself. Using the "cell factory", select different host systems and production methods to product recombinant spider silk protein has become the mainstream method at these days.Due to the excellent strength of dragline silk protein MaSp1, it attracts much attention. It is also the main component of dragline silk protein MaSp2 which did not get much attention, but it still has excellent elastic properties. The main technical difficulties at the present stage of synthesis of recombinant spider silk protein are synthesis of large molecular weight genes and expression of high molecular weight protein.This paper used the methods of genetic engineering and synthetic biology, and synthesis about 10000 bp molecular weight of the MaSp2 gene by seamless splicing technology. Regulate the supplyment of amino acid tRNA in the protein translation process. And make it is possible to product high molecular weight spider silk protein. The major breakthrough in the subject is expressed the molecular weight of 110 kDa MaSp2 protein.The main results are as follows.(1) Successed to synthesised the MaSp2 protein gene of Nephila clavipe. Comparative and analysis the repeat region of MaSp2 protein amino acid sequence. And get a high degree of repeat sequence to synthesis. Using the same enzyme tail seamless splicing technology to get 2,4,8,16,32,64,96 multimers.(2) Cloned the spliced gene framents into the expression vetor pET28a to construct the expression cassette and expressed. And we successfully obtained the molecular weight of 55 kDa recombinant spider silk protein MaSp2 and yield can reach 20 mg·L-1.(3) We overexpression the alanine tRNA genes alaT and glycine tRNA genes glyVXY by increasing the gene copy number. And through this method we get the molecular weight of 110 kDa MaSp2 protein.(4) We through the high-density fermentation technology successfully obtained 150 mg·L-1 protein yield of recombinant protein MaSp2. And explore the spinning method of it.The innovation of this paper is that using the same tail gene seamless splicing technology and synthesized large molecular weight gene fragment of MaSp2 in vitro. And success to express the 110 kDa molecular weight of the MaSp2 recombinant protein by regulating the content of key amino acid tRNA gene. This is also the highest molecular weight MaSp2 protein by synthetic. And open up a new way to study of protein MaSp2.