Expression of a fusion protein containing calf prochymosin B(1-161) and human proinsulin.
The plasmid pJG202 containing Tac promoter, calf prochymosin B(1-161) gene and human proinsulin gene was constructed and transformed into E. coli JM105. The expression of the fusion protein consisting of 249 amino acids was controlled by IPTG and temperature, and the expressed protein was estimated to be 20-35% of the total cellular proteins by scanning of the SDS-PAGE gel stained with Coomassie brilliant blue R250. After CNBr cleavage, sulfitolysis, partial separation of the S-sulfonated human proinsulin and recombination of the disulfide bonds, human proinsulin with native properties, as evidenced by amino acid composition analysis, receptor binding and radioimmunoassays, could be obtained.
Tang
J
J
National Laboratory for Protein Engineering and Plant Genetic Engineering, Peking University, Beijing, China.
Xue
Y
Y
Fan
X
X
Fu
Y
Y
D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011384:Proinsulin / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011384:Proinsulin; C032443:prorennin; D012085:Chymosin