Bio-Engineering of Human
Insulin
Bio-Engineering of Human
Insulin
Bio-active
Insulin comprises two polypeptides, Chain A and Chain B, transcribed from a
single gene locus, and translated as preproinsulin. In
vivo, preproinsulin is modified to remove first a
Signal Peptide, producing proinsulin, and then
the intervening C-chain. The A and B chains
then self-assemble. A more efficient in vitro process
would avoid post-translational processing, and synthesize Chain A and Chain B directly.
(Line 1) An Expression
Vector is a plasmid with a selectable marker AmpR
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 NH2-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 together by three disulfide bonds, two between
the A & B chains, and one between
residues in the A chain.