Bio-Engineering of Human Insulin

    Bio-active Insulin comprises two polypeptides, Chain A and Chain B, transcribed and translated from the same gene locus. (Line 1) An Expression Vector is a plasmid with a selectable marker Amp^R for ampicillin resistance, and the promoter and structural gene for B-galactosidase. Each DNA is cloned into the 3' end of the structural gene. (Line 2) The plasmid is transformed into an E. coli host. Upon induction with B-galactose, the promoter directs transcription and translation of a fusion protein comprising the B-galactosidase and Insulin chain. (Line 3) This reaction is carried out in bulk culture, or at industrial scale in a bio-reactor, an industrial-scale chemostat that maintains cell multiplication at a constant optimum rate, while cells are harvested for processing. (Line 4) Cyanogen Bromide (CnBr) cleaves the insulin polypeptide at its initial 3'-methionine from the fusion protein. (Line 5) When purified separately and combined in vitro, Chain A and Chain B spontaneous assemble into bio-active Insulin, the tertiary structure held togehther by three disulfide bonds, two between the A & B chains, and one between residues in the A chain.