Conjunction Of Spider Silk Protein By Trans-Splicing Of Split-Intein

Spider silk is an ideal system for studying the relationship between protein design and function, because of their superiority in characters such as high elasticity, extreme toughness and extensibility. Despite significant interest in other silk sources, such as spider silk, silkworms have been the traditional source exploited. One reason that spider silk has lagged behind is that silkworms are fairly easy to domesticate, whereas spiders can not be housed in high densities. Therefore, the best option for moving the application of spider silks forward is the pursuit of biotechnological means of generating source material. The length of the spider gene and the high repeat domain in spider protein are the main obstructs in the road of production of spider silk by genetic engineering.Intein is a protein-intervening sequence, and its flanking sequences are called extein. By four nucleophilic displacement reactions, intein catalyzes exteins to ligate with a peptide bond to produce the mature host protein, with itself precisely excised from precursor protein in the post-translational maturation process. This intein mediated unique autocatalytic biochemical reaction provides us a new method for protein synthesis and modification. The discovery of the split-intein gives a high light on the protein engineering, because it can ligate the interested protein in vitro. There is a wide perspective for further applications of intein-based protein engineering.In this study, two split inteins which showed high trans-splicing efficiency were used to ligate the spider silk protein in vitro. A～700bp partial fragment of spider flagelliform repeat domain was isolated from Araneus ventricosus gene library and this fragment contain an intact repeat domain of flagelliform silk gene. The gene was then optimized and repeated seven times, meanwhile added the non-repeat N terminal anc C terminal domains to the flank of the repeat fragments. The longer silk gene was then broken into three fragments and inserted into the middle of two inteins fragments to construct three sandwich expression vectors. The final expression plasmids, pE-N-Sc2-SG, pE-SG-Sc3-RB, pE-RB-Sc2-C, were induced to express the recombinant protein by IPTG and then purified the three recombinant silk proteins separately. The purified proteins can be ligated by the two inteins between the silk proteins. In this study, the efficiency of once splicing is about 60% to 80%, and proportion of twice splicing products is about 20%. This is the first time to get so large silk protein (twice products), the molecular weight of which is about 150 kD. The prokaryote expressing system has many advantages, such as low production cost and convenience for large-scale operation in industrial production of the silk protein, and at the same time this ligation system can be applied into production of other complicated proteins.