Construction Of Genetic Engineering Strain Expressing Fusion Protein HSA-CP

C-peptide(CP), a linking chain with 31 amino acids in length, locates between A and B chain of insulin. The recent researches indicate that it elicits a number of cellular responses by specific binding to G-protein coupled receptor. Including increasing Ca2+ influx, stimulation of Na+-K+-ATPase and endothelial NO synthase activities resulting in the increase of blood flow and expansion of blood vessel. Therefore, CP can ameliorate sensory nerve dysfunction, nephropathy, neuropathy and microvascular dysfunction. In order to prolong the half-life of CP in vivo, we developed a potential long-acting CP(HSA-CP) by albumin fusion technology. The fused gene HSA-CP was expressed in pichia pastoris. Confirmed by Western blot and cell proliferation analysis, the fusion protein was purified preliminarily.To improve the expression of HSA-CP, the codon usage of the gene encoding for CP was optimized according to Pichia pastoris codon bias and reduced repetitive AT and GC content. The artificial CP gene was fused to the HSA cDNA in the same reading frame and then sequenced. The recombinant plasmid named pBlue-HSA-CP. The fused gene HSA-CP was recovered from pBlue-HSA-CP digested with DNA restriction enzyme EcoRâ… and Notâ… , and then inserted into expression vector pPIC9k to construct recombinant plasmid pPIC9k-HSA-CP. The results showed that the cloned fused gene was in correspondence with our anticipation, and expression vector pPIC9k-HSA-CP was constructed correctly.The recombinant vector pPIC9k-HSA-CP was linearized by Salâ… , then transformed into Pichia pastoris strain GS115 by electroporation. The Mut+ transformants were selected out from histidine-deficient medium plates and screening for Mut phenotype. Confirmed integration by PCR, The recombinant strains were induced to express fusion protein HSA-CP by methanol in shake. The genetic engineering Pichia pastoris whose expression level was relative high was identified by western bolt and cell proliferation analysis. After optimized the inducing time and concentration of inducer, the secreted fusion protein HSA-CP was purified. Finally, 8 strains were screened out from contained 3 mg/mL G418 MD plate, in which two high expression strain, named as rHC1, rHC2 could secrete 120 mg/L fusion protein was obtained with culturation in shakes. The expressed fusion protein has apparent molecular weight of 70 kDa observed by SDS-PAGE. It was specifically recognized by an anti-human HSA polyclonal antibody and CP polyclonal antibody in Western blot assay. After optimized the culture condition, the yield of the recombinant strain was raised up to 140mg/L determined by Microalbuminuria Immunoassay kit and C-peptide Chemilumin escence Immunoassay kit. Further studies on its cell activites, we found that C-peptide and HSA-CP in low concentration can effect on 293 cell proliferation.