Recombinant Human Insulin

RECOMBINANT  HUMAN INSULIN

Recombinant human insulin, used for the treatment of diabetes, was the first drug produced using genetic engineering, in 1982. Before this, animals, notably pigs and cattle, were the only non-human sources of insulin. Animal insulin differs slightly from the human form and, consequently, it can potentially elicit an immune response in humans, making it ineffective. Additionally, the use of recombinant insulin prevents the risks resulting from potential contamination of animal insulin with other hormones or viruses. To understand how insulin is produced using recombinant DNA techniques we first need to review its structure. Figure 25.8 illustrates how insulin is initially synthesized as preproinsulin, a single polypeptide which is processed during export into proinsulin and finally into active insulin, once proteolytic cleavage of the connecting sequence for the two A and B insulin chains has occurred. These two chains remain joined through disulphide bonds.

Currently, different approaches are employed to produce recombinant insulin, one of which is shown in Figure 25.8. First of all, two DNA fragments coding for the A or the B insulin chains are synthesized chemically.

Figure 25.8 Production of recombinant insulin. (A) Insulin consists of two polypeptide chains (chains A and B). It is initially synthesized as part of a larger peptide called preproinsulin . The transport across the cell membrane of preproinsulin results in the cleavage of the signal peptide and the formation of disulphide bridges to generate proinsulin . Finally, the connecting peptide is cleaved generating the mature insulin. (B) One of the strategies used to make recombinant insulin consists of the cloning of the DNA fragment coding for the A chain and the B chain into two separate expression vectors as β - galactosidase fusions in E. coli . The fusion proteins are then purifi ed and the insulin is cleaved with cyanogen bromide (CNBr) after a methionine incorporated at the β - galactosidase and insulin junction. The presence of several methionines in the β - galactosidase results in multiple cleavage of this molecule by CNBr. Finally, the resulting insulin A and B chains are refolded and the cysteines are oxidized for the generation of the active insulin.  

Each of these synthetic fragments is then individually inserted in a plasmid after the E. coli gene coding for β galactosidase. This enables this bacterium to produce large fusion proteins with the insulin chains tacked on to the end of the β-galactosidase enzyme. These fusion proteins can then be purified from bacterial extracts and the insulin chains released upon treatment with cyanogen bromide, which cleaves peptide bonds following methionine residues. As methionine was inserted at the boundaries between the β-galactosidase and the insulin chains, and there are no methionines present internally within the insulin molecule, treatment with cyanogen bromide results in the cleavage of intact insulin chains from the fusion proteins. The purified A and B insulin chains can be mixed and reconstituted into an active insulin molecule. Currently there are also other methods used to produce recombinant insulin which are based on the generation of single β-galactosidase fusions to the full-length insulin gene containing the coding sequences for both A and B chains. These alternative methods can simplify the manufacturing of this drug.