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A new method for the preparation of human parathyroid hormone 1–34 peptides
Author(s) -
Xiu Zhaoyang,
Li Min,
Zhou Suijing,
Dou Hong,
Zhou Heyue,
Chen Changqing
Publication year - 2002
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20020015
Subject(s) - recombinant dna , affinity chromatography , escherichia coli , biochemistry , chemistry , fusion protein , parathyroid hormone , microbiology and biotechnology , sepharose , biology , chromatography , enzyme , gene , calcium , organic chemistry
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.