[Studies on genetic engineering of human insulin-purification and characterization of human proinsulin and insulin].
E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.
Guo
L H
LH
Shanghai Institute of Cell Biology, Academia Sinica.
Zhou
M Y
MY
Shen
M H
MH
Wang
E B
EB
Liu
J F
JF
Yu
Y J
YJ
D003001:Cloning, Molecular; D004926:Escherichia coli; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007328:Insulin / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics*; D011384:Proinsulin / Q000235:genetics / Q000302:isolation & purification*
D007328:Insulin; D011384:Proinsulin