Synthesis Of Spider Silk-protein Gene And Recombinant Expression Of Its Multimers

Spider silks,being a class of nature protein fibers,can be widely used in the biomedical,material,textile and military areas,because of their unique mechanical features of high strength,elasticity and high energy to break,and eminent biological peculiarities in terms of biodegradability and compatibility to the biological tissues.Because of inevitable problems in spider cultivation to produce natural spider silks, large amount of spider silks for their applications can only be obtained through genetic engineering.In this paper,an genetic spider silk protein unit was designed on the basis of amino acid sequences which is previously reported,in which the features of structural modules and their functional correlations were particularly concerned.Theoretically,this new-type spider silk protein is particularly endowed with two main pecaularities of spider silks which are high-intensity and high-elaticsity.The gene SPS(405 bp)encoding this silk protein unit was delicately synthesized by the manners of overlapped primer extension, finally verified by DNA sequencing.SPS gene was fused behind the tag of thioredoxin(TrxA)in plasmid pET-32a(+)to generate the expression vector pET-SPS1.This vector was further used as the backbone for creation of a set of expression vectors,pET-SPS(2～8)having different multimers of SPS gene by means of isocaudarners,all SPS vectors were expressed in E.coli strain, BL21(DE3)by IPTG induction,and the expression of fusion proteins of spider silk were purified by His-tag affinity chromatography.All SPS fusion proteins expressed in E.coli are almost fully soluble.The vectors with 1-3 multimers of SPS could be efficiently expressed,while more increased multimers could only cause worse expression.In addition,plant host was used in this work to produce recombinant spider silk proteins.For that,a few of SPS plant expression vectors of pBI-SPS(5～7)were generated,and transformed into Agrobacterium strain LBA 4404 by freeze-thaw method. These engineered strains were subsequently transformed by tobacco plants leaf disc infiltration.Finally,several corresponded lines of regenerated tobacco plants were obtained through tissue culturing with strict antibiotic selection.In one word,this work was completed with the success of gaining the gene encoding a new- type of artificial spider silk protein with distinct features of high intensity and elasticity.Prelimilarily expression of this gene and its mutimers were investigated by using the systems of E.coli and plants,which could Laying the foundation of applications of this new-type silk protein in the future.