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Biotechnology and Applied Biochemistry (2002) 36, (111–117) (Printed in Great Britain)

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A new method for the preparation of human parathyroid hormone 1–34 peptides

Zhaoyang Xiu, Min Li, Suijing Zhou, Hong Dou, Heyue Zhou and Changqing Chen¹

Research Center of Biotechnology, Shanghai Institute for Biological Sciences, The Chinese Academy of Science, Shanghai 200233, China

Key words: adenylate cyclase, anhydrochemotrypsin, affinity purification, fusion expression, prolyl endopeptidase (PEP).

Abbreviations used: GST, glutathione S-transferase; PEP, prolyl endopeptidase; hPTH, human parathyroid hormone; IPTG, isopropyl b-D-galactoside; Nle, norleucine.

¹To whom correspondence should be addressed (e-mail cqchen@srcb.ac.cn).

An engineered Escherichia coli strain, BL21 (DE3)/pGEX-4T-human parathyroid hormone (hPTH) (1–34), was constructed by oligonucleotide annealing and PCR amplification of the target gene, and then by ligating it with the pGEX-4T-3 vector and transferring into the BL21 host. The soluble glutathione S-transferase (GST) fusion protein GST-hPTH (1–34), expressed from BL21 (DE3)/pGEX-4T-hPTH (1–34), was harvested after fermentation and purification by affinity chromatography. Following double cleavage by thrombin and prolyl endopeptidase, about 0.6 g/l intact hPTH (1–34) was harvested. The product was checked by HPLC MS and N-terminal sequence analysis. The purified recombinant hPTH (1–34) stimulates adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic hPTH standards, indicating that the recombinant product has full biological activity.

Received 11 March 2002/27 April 2002; accepted 9 May 2002

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