Construction And Secretive Expression Of RGD-Spider Dragline Gene In Pichia Pastoris

The study on spider silk,especially dragline,as a high-performance biomaterial has aroused attention.The expression of recombinant dragline protein with RGD motif has been accomplished in E.coli.in our previous work.DNA monomer was redesigned and synthesized with codons bias of Pichia pastoris.Two restriction sites were located in the end of DNA monomer,which was used for the cloning and multimerizing of the monomer,respectively.DNA monomer was cloned to vector- pBluescript SKⅡ.Then,the DNA monomer was used to construct multimers by the "the head to tail" strategy after sequenced.Several genetically engineered strains were obtained,such as 2-multimer strains,4-multimer strains,8-multimer strains,16-multimer strains,32-multimer strains.Each of 16-multimer and 32-multimer spider dragline gene was cloned to shuttle expression vector- pPIC9K,thus the contructs YNSR-16 and YNSR-32 were obtained.The linized contructs was transformed to Pichia pastoris host strain GS115,then,recombinant strains were acquired, respectively.Stable multicopy recombinant Pichia pastoris were selected by various G418 concentrations.Recombinants of multicopy inserts were identified by PCR(a-factor,3'AOX1 primer).The result indicated that the target gene had been integrated into the genome of GS115. The construction of multicopy engineered strains was successful.The product of 16-multimer recombinants were analysed by SDS-PAGE.The results demonstrated that recombinant dragline protein was secreted by 16-multimer recombinants,of which the molecular weight was 47KD.The shake flasks methods was established as follows: the medium pH-6.0,the culture temperature-30℃,the concentration of methanol-1.5%,the inducing time-96h.The obtained 16-multimer and 32-multimer recombinants were used.We investigated the effect of the properties and integrant sites of recombinant dragline protein genes on the expression of recombinant spider dragline silk protein.Because of high repetitive sequence and GC content,recombinant dragline protein genes were transcript into mRNA with complex secondary structure.This led to reduced expression of recombinant dragline protein.The target genes were integrated to different sites in host genome.We concluded that the recombinant dragline protein was secreted as 16-mer gene intergrated to His4 gene in genome.