Construction, expression and characterization of recombinant fusion protein HSA-PTH (1-34) in Pichia pastoris / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.</p><p><b>METHODS</b>HSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.</p><p><b>RESULT</b>The PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).</p><p><b>CONCLUSION</b>Active fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.</p>