[Construction, expression and characterization of recombinant fusion protein HSA-PTH (1-34) in Pichia pastoris].
To obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.



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.



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).



Active fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.
Chen
Jing
J
Department of Biochemical Pharmacy, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.
Sun
Hong-ying
HY
Yang
Ying
Y
Wang
Xue-fen
XF
Chen
Shu-qing
SQ
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics; D019379:Teriparatide / Q000031:analogs & derivatives*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D019379:Teriparatide; C035748:parathyroid hormone (1-34)amide