The Biosynthesis Of Spider Dragline Silk Protein Containing The RGD Motif And Preparation Of Silk Film

In this paper, the spider dragline silk with the most remarkable mechanical properties was investigated as high-performance biomaterials. The gene of the spider dragline silk proteins with the introduced RGD motif which involves the cell adhesion, was designed and expressed by the genetic engineering technology. Large quantities of recombinant proteins were gained by the high-density fermentation of recombinants and purification, then the purified RGD-spider dragline proteins were developed as the novel membrane biomaterials, applied to the tissue engineering. The work in this paper includes the following:1 The monomer repeat of spider dragline silk protein was designed with the RGD motif introduced into the monomer sequence which involves the cell adhesion, and the gene encoding for the monomer repeat was synthesized by chemistry Then the monomer repeat was used to construct the multimers by the "the head to tail" strategy. The repeat unit was doubled after every step of constructing. The obtained 8-multimer and 16-multimer recombinants were named as pSDR-8 and pSDR-16 respectively.2 The pSDR-8 and pSDR-16 were digested with BamHI/HindIII doubly , and the released fragments of the 8-multimer and 16-multimer were purified and cloned into the prokaryotic expressing vector pET-30a(+) , and transformed into the host of E.coli Bl21(DE3)pLyS, the expressing recombinants were named as pNSR-8 and pNSR-16 respectively. The expression of recombinant protein was induced in the prokaryotic expressing system with high effectiveness by the addition of IPTG.3 The expressed products were purified through His-Bind resin ,and submitted to the SDS-PAGE analysis ,the result shows that the molecular weight oftwo products expressed is 35kD and 60kD respectively, in agreement with the desired values. S.tag Western Blot confirms the specification of products.4 The Isoelectric point of the products of pNSR-16 was determined by Gel Isoelectric Focusing, and the value is pI6.4. Large quantities of pNSR-16 were obtained through the high-density fermentation , and large scale of recombinant spider silk protein was purified according to the its chemical and physical properties .5 The synthetic spider silk protein was dissolved in the formic acid to prepare the silk protein solution with the concentration up to 15%.The solution was scraped on the polyethylene disk, and the methods for preparing the silk film with the recombinant spider silk proteins were discussed: the protein solution on the disk was dried at 37C, then transferred into the ethanol solution for protein denature and then dried at 31 C again or was dried at the -60 C in the vacuum after frozen at -70 C. The silk film of final formation which has the resistance to the water was obtained.