Recombinant insulin Production

Recombinant insulin Production

Insulin, synthesized by the β-cells of the islets of Langerhans in the pancreas, controls the level of glucose in the blood.

An insulin deficiency manifests itself as diabetes mellitus, a complex of symptoms which may lead to death if untreated. 

Insulin used in this treatment was originally obtained from the pancreas of pigs and cows.

Problems of animal-derived Insulin

1. Side effects in some patients
2. Purification procedures are difficult
3. Potentially dangerous contaminants cannot always be completely removed

Features that facilitate its production by recombinant DNA techniques - 

1. Human protein is not modified after translation by the addition of sugar molecules
2. Size of the molecule is small, comprising two polypeptides, one of 21 amino acids (the A chain) and one of 30 amino acids (B chain)

Preproinsulin precursor which contains the A and B segments linked by a third chain (C) and preceded by a leader sequence.

Method -

1. The leader sequence is removed after translation and the C chain excised, leaving the A and B polypeptides linked to each other by two disulphide bonds.
2. Then the artificial gene was ligated to a lacZ′ reading frame present in a pBR322-type vector of E. coli.
3. The insulin genes were expressed as fusion proteins, consisting of the first few amino acids of β-galactosidase followed by the A or B polypeptides which were separated by a methionine residue.
4. Then insulin polypeptides cleaved from the β-galactosidase segments by treatment with cyanogen bromide.
5. Then purify the A chains & B chains then finally carry-out disulphide bond formation