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]