How human insulin is manufactured

Genentech was the first company to produce insulin through recombinant DNA technology in E coli. The genes for human
insulin A and B chains were cloned separately (10 pts) as fusion proteins to β-galactosidase (5 pts) (at the carboxy-terminus),
cleaved from β-galactosidase using cyanogen bromide, and then disulfide bonds were added in vitro (10 pts) (ref 2). The
procedure of expressing the insulin was split into five main parts: (i) Assembly of insulin genes, (ii) Construction and
characterization of lac-insulin hybrid plasmids, (iii) Preparation of insulin reagents, (iv) Purification of the B chain of human
insulin, and (v) Purification of the A chain of human insulin. Plasmid pBR322 was isolated for the recombinant DNA
expression procedure. Both the A and B insulin chain genes were assembled using phosphorylation of oligonucleotides and T4
polynucleotide kinase. The phosphorylated fragments of DNA were ligated. The B gene was then purified using gel
electrophoresis where the slowest band of DNA was extracted. The same thing was done with the gene that coded for the A
chain of human insulin. The A and B chain genes were then individually excised from pBR322 using the restriction enzymes
BamHI and HindIII. The A chain gene was inserted into the insulin expression plasmid pIA1 and the B chain gene was inserted
into pIB1. pIA1 was introduced into E. coli K-12 strain 294 whereas pIB1 was introduced into E. coli K-12 strain D1210.
During mass production of the insulin chains, insulin reagents were constructing for the post translational modification of both
chains. S-sulfonated derivatives of the A and B insulin chains were isolated from porcine and bovine insulin. The S-sulfonated
derivatives are the key to form the three disulfide bonds and connect the chains together. Each strain was isolated by lysing the
cell and centrifugation to get rid of the cell debris, and pH variance and centrifugation to isolate the protein. The S-sulfonated
derivatives were then prepared to properly combine and fold A and B chains to create human insulin.
The two issues that are brought up in trying to express insulin in E. coli include: getting rid of the C protein chain when E. coli
does not have the appropriate proteases, and adding the disulfide bonds when Cysteine is not available to create the bonds. The