Gene segments
corresponding to the mature
A
& B chains of insulin are engineered into
separate
bacterial plasmids with
an antibiotic resistance
gene and a B-gal
structural
gene as markers.
The
plasmids are separately transformed
into E. coli.
Culture of the E. coli
in the
presence of antibiotics selects those
cells that have been successfully transformed. Induction of
the B-gal
gene
expression (by addition of B-galactose
sugar) also causes transcription of the insulin genes, which are then
translated by the bacterial cells.
The
bacterial
culture is done on an industrial scale to produce thousands of
litres of cloned human insulin.
Following chemical cleavage and purification away from the plasmid
protein,
the A & B chains spontaneously assemble to
form
biologically-active
insulin. As this
processin vitro is relatively inefficient, commercial
human
insulin is now made by cloning the proinsulin
mRNA (without the signal peptide of pre-proinsulin) in a single host, and
specific cleavage with the proteolytic enzymes.
