title: Enterokinase cleavage of fusion proteins for preparation of recombinant human parathyroid hormone 1-34.
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.
authors.affiliation: Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Science, Shanghai 200233, China.
authors.forename: Zhao-Yang
authors.initials: ZY
authors.lastname: Xiu
authors.forename: He-Yue
authors.initials: HY
authors.lastname: Zhou
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.forename: Jin-Feng
authors.initials: JF
authors.lastname: Dai
authors.forename: Chang-Qing
authors.initials: CQ
authors.lastname: Chen
mesh_terms: 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
chemical_list: D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004765:Enteropeptidase; D000262:Adenylyl Cyclases