Enterokinase Cleavage of Fusion Proteins for Preparation of Recombinant Human Parathyroid Hormone 1—34

XIU Zhao-Yang, ZHOU He-Yue, YU Ying, DAI Jin-Feng, CHEN Chang-Qing^*
( Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Science, Shanghai 200233, China )

Abstract    An engineering E.coli strain, BL21 (DE3)/pGEX-4T hPTH (1—34), was constructed by oligonucleotide annealing and PCR amplifying the target gene, then ligating it with pGEX-4T-3 vector and transferring into BL21 host. The yield of soluble fusion protein of GST-hPTH(1—34) expressed from BL21(DE3)/pGEX-4T hPTH(1—34) is about 10 g/L after high-density, high expression culture and purification by affinity chromatography. Following the simple digestion of enterokinase, about 0.6 g/L intact hPTH (1—34) was harvested. The product is checked by HPLC、MS and N-terminus sequence analysis. The purified recombinant hPTH(1—34) stimulated adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic human parathyroid hormone standards, indicating that the recombinant product has full biological activity.
Key words    parathyroid hormone; enterokinase; anhydrochemotrypsin affinity resin; affinity purification

^*Corresponding author: Tel, 86-21-64700892-306; Fax, 86-21-64700244; e-mail, [email protected]