Eucaryotic Expression And Bioactivity Evaluation Of Fused Protein HPTH(1-34)_ab-HSA

Human Parathyroid hormone (hPTH) is one of the most important hormones that could regulate bone metabolism. It is secreted by the principal cells of parathyroid gland and has a single strand who contains 84 amino acid residues. Its analogs have been exploited into a new series of drugs to treat osteoporosis. Study shows that the bioactivity of the native hPTH could be retained completely by the first 34 amino acids and the half-life of small-molecule-proteins can be extended if it been fused to Human Serum Albumin (HSA). In order to raise the bioactivity of hPTH and prolong its half-life in vivo, we constructed the concatenate of hPTH(1-34) and then fused it to HSA. The fused gene was expressed in Pichia.pastoris. After gaining the fused protein hPTH(1-34)_ab-HSA, we confirmed, purified it and evaluated its bioactivity preliminarily.Two kinds of hPTH(1-34) genes that with different 5′and 3′tails were amplified by two pairs of primers that with high specificity and then spliced by overlapping PCR. The concatenate gene was cloned into plasmid pTG19-T and then sequenced the recombinant plasmid pTG19-T-hPTH(1-34)_ab. The result demonstrated that the nucleotides sequence of cloned concatenate gene was completely consistent with the one we expected. Spliced the concatenate hPTH(1-34)_ab and HSA genes in vitro by overlapping PCR. The fused gene we gained was inserted into expression vector pPIC9K at EcoRⅠand NotⅠrestricted endonuclease sites, and then identified and sequenced the recombinant expression plasmid pPIC9K-hPTH(1-34)_ab-HSA. Identification by double enzyme digestion showed that the recombinant expression plasmid had been constructed successfully. The result of sequencing demonstrated that the nucleotides sequence of cloned fused gene was in correspondence with our anticipation.The recombinant expression plasmid pPIC9K-hPTH(1-34)_ab-HSA linearized at SalⅠrestricted endonuclease site was transformed into Pichia.pastoris GS115 by electroporation. We screened out recombinant strains with relatively high expression level by G418 resistance and expression level by step to step. Then identified their production by Western blotting and optimized their expressing conditions. At last, 5 strains were screened out from the MD plate which containes 2 mg/mL G418. Two strains among them could express fused protein hPTH(1-34)_ab-HSA in relatively high level. Western blotting analysis with polyclonal antibody of HSA and PTH indicated that the antigenicity of the fused protein was specific. The maximal yield of fused protein could reach 122.8 mg/L.Purified the fused protein and analyzed its bioactivity preliminarily by measuring the RANKL, OPG and IGF-1 mRNA expression in UAMS32pB cell line. The conclusion is that we can gain the fused protein with electrophoresis purity by ultrafiltration and affinity chromatography. Preliminary analysis of bioactivity indicated that hPTH(1-34)_ab-HSA has better osteogenic activity than hPTH(1-84)-HSA.