A new method for the preparation of human parathyroid hormone 1-34 peptides.
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.
Xiu
Zhaoyang
Z
Research Center of Biotechnology, Shanghai Institute for Biological Sciences, The Chinese Academy of Science, Shanghai 200233, China.
Li
Min
M
Zhou
Suijing
S
Dou
Hong
H
Zhou
Heyue
H
Chen
Changqing
C
D000262:Adenylyl Cyclases / Q000096:biosynthesis; D000818:Animals; D002460:Cell Line; D019020:Directed Molecular Evolution / Q000379:methods*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007672:Kidney Cortex / Q000187:drug effects / Q000201:enzymology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019379:Teriparatide / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D019379:Teriparatide; D000262:Adenylyl Cyclases