Expression, Purification, Identification Of Fusion Protein PTD-SARA

Introduction Overwhelming evidence shows that transforming growth factor-β1(TGF-β1)plays a central role in the pathogenesis of renal fibrosis. The activated TGF-β1 binds to its receptors and mediates renal fibrosis via the downstream Smad signaling pathway.Smad anchor for receptor activation (SARA)protein plays important roles in the fibrotic promoting signaling pathway initiated by TGF-β1.Its specific binding to Smad2 and Smad3 through the Smad-binding domain of SARA(SARA-SBD,amino acids 665-750 of SARA)inhibits Smad phosphorylation,consequently inhibits the specific interaction of Smad2 and Smad3 with Smad4 and accordingly prevents the process of fibrosis.Genetic engineering strategies can provide a solution to express fusion protein PTD-SARA in E.coli,which can block the binding of smad2,smad3 with Smad4,once enter the cells,and thus effectively alleviate the renal fibrosis.Objective To express a recombinant fusion protein PTD-SARA in E.coli and acquire the purified protein.Methods The full-length PTD-SARA gene has been synthesized using the E.coli preferred codon.This gene was cloned into vector pQE30,and then the recombinant plasmid was transformed to E.coli Ml5 and induced the expression with IPTG.The expressed fusion protein was purified by Ni²⁺-NTA column chromatography and identified by both Western blot analysis and the N-terminal amino acid sequencing.RESULTS The recombinant plasmid pQE-30-PTD-SARA was constructed successfully.The enzyme digestion and the sequencing results showed that the target gene was inserted in the right orientation.The PTD-SARA protein was stably expressed by using the constructed recombinant plasmid pQE-30-PTD-SARA.After purification,the purity of the target protein could be as high as 90%or more.CONCLUSION The methods for construction, expression and purification of recombinant PTD-SARA have been established, which are essencial for the research of protein function in vitro and in vivo.