Question

Some of the steps involved in the production of humulin are given below. Choose the correct sequence.
I. Synthesis of gene (DNA) for human insulin artificially
II. Culturing recombinant E.coli in bioreactors
III. Purification of humulin
IV. Insertion of human insulin gene into plasmid
V. Introduction of recombinant plasmid into E.coli
VI. Extraction of recombinant gene product from E.coli

• A. I, IV, V, II, VI, III
• B. III, V, II, I, VI, IV
• C. II, I, IV, III, V, VI
• D. I, III, V, VI, II, IV

Answer 1

The correct option is A I, IV, V, II, VI, III

The first step of manufacturing humulin is to chemically synthesise the DNA chains that carry the specific nucleotide sequences characterising the A and B polypeptide chains of insulin. The required DNA sequence can be determined because the amino acid compositions of both chains have been charted. Sixty three nucleotides are required for synthesising the A chain and ninety for the B chain, plus a codon at the end of each chain,signalling the termination of protein synthesis. An anti-codon, incorporating the amino acid, methionine, is then placed at the beginning of each chain, which allows the removal of the insulin protein from the bacterial cell's amino acids. The synthetic A and B chain genes are then separately inserted into the gene for a bacterial enzyme, B-galactosidase, which is carried in the vector's plasmid. At this stage, it is crucial to ensure that the codons of the synthetic gene are compatible with those of the B-galactosidase. The recombinant plasmids are then introduced into E. coli cells. The protein which is formed, consists partly of B-galactosidase, joined to either the A or B chain of insulin. The A and B chains are then extracted from the B-galactosidase fragment and purified. The two chains are mixed and reconnected in a reaction that forms the disulfide cross bridges, resulting in pure humulin, synthetic human insulin.