February 9, 2011

insulin production - APPSC g1 mains

This peptide hormone i.e. insulin is secreted by the Islets of Langerhans of pancreas which catabolizes glucose in blood. Insulin is a boon for the diabetics whose normal function for sugar metabolism generally fails.

Insulin consists of two polypeptide chains, chain A (21 amino acid long) and B (30 amino acid long). Its precursor is proinsulin which also contains two polypeptide chains, A and B, and is connected with a third peptide chain-C (35 amino acid long). However, the recent discoveries reveal that precursor of insulin is pre- pro insulin which is about 109 amino acids long. The pre-pro- insulin is synthesized in beta cells of pancreas, the structure of which is given below :

NH2-(Peptide)-b-Chain- (peptide-c)- A chain-COOH

In the beginning, efforts were made to isolate mRNA for pre- pro- insulin from rats Islets of Langerhans of pancreas and to synthesize cDNA. Thereafter, it was inserted into a plasmid. The recombinant plasmids were transferred into the E. coli cells which secreted pro-insulin.

Itakura et. al. (1977) chemically synthesized DNA sequence for two chains, A and B, of insulin and separately inserted into two pBR322 plasmids by the side of b-galactosidase gene. The recombinant plasmids were separately transferred into E coli cells which secreted fused b-galactosidase - A chain and b- galactosidase - B chain separately. These chains were isolated by detaching from b-glactosidase in pure form in a amount of about 10 mg/24 g of healthy and transformed cells (Sasson, 1984).

Detachment of proinsulin could be possible when an extra methionine codon was added at the N-terminus of each gene for A and B chains. The two chains (A and B) were joined in vitro to reconstitute the native insulin by sulphonating the two peptides with sodium disulphonate and sodium sulphite. Gilbert and Villakomaroff (1980) isolated mRNA for insulin from B cells of rat's pancreas and inserted into pBR322 plasmid in the middle of a gene normally coding the penicillinase, and incorporated it into E. coli cells. E. coli cells produced a hybrid protein (penicillinase+proinsulin) from which the proinsulin was separated by using trypsin. It is estimated that clones of E. coli are capable of producing about one million molecules of insulin per bacterial cell. Human insulin (humulin) is the first therapeutic product produced by means of recombinant technology by Eli Lilly & Co.


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