Enterokinase cleavage of fusion proteins for preparation of recombinant human parathyroid hormone 1-34.
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.
Xiu
Zhao-Yang
ZY
Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Science, Shanghai 200233, China.
Zhou
He-Yue
HY
Yu
Ying
Y
Dai
Jin-Feng
JF
Chen
Chang-Qing
CQ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004765:Enteropeptidase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004765:Enteropeptidase; D000262:Adenylyl Cyclases