1. Synthesis of Polynucleotide Chains: Insulin molecules are composed of two polynucleotide chains. A chain and B chain. A chain consists of 21 and B chain of 30 amino acids. A chain of 63 nucleotides and B chain of 90 nucleotides are made chemically in the laboratory. Then the two chains are purified.
2. Production of recombinant DNA: Carrier bacteria are selected. Plasmid DNA is isolated from bacterial cells. Restriction enzymes are used to remove certain parts from A chain and B chain. Again, corresponding segments are cut from plasmid DNA using the same enzyme. However, the portion of the plasmid DNA that produces the gene for the β-galactosidase enzyme is cut out. The A chain and B chain are then joined to separate plasmid DNA with the help of ligase enzyme. Recombinant DNA is formed as a result of ligation of A chain and B chain with plasmid DNA. Thus two types of recombinant DNA are produced. A strand is recombinant DNA and B strand is recombinant DNA.
3. Introduction of Recombinant DNA into Host: Recombinant DNA is introduced into host bacterial cells during the transformation process. This is done by heat-shock method or electric pulse method. The host (E. coli) is first placed in CaCl2 solution and then on ice for 14-16 hours. It binds Ca to the host cell wall. A mixture of host and recombinant DNA in a container was placed on ice for 30 min, at 420°C for 90 s, and again on ice for 2 min. In this the host (E. coli) absorbs the recombinant DNA.
4. Gene Cloning: Host bacteria (E. coli) with recombinant DNA are grown in culture medium in fermentation tanks. As the number of E. coli bacteria increases, recombinant DNA copies are also produced. This results in β-galactosidase A chain and β-galactosidase B chain. Also some methionine is produced.
5. Separation of the two chains: A chain and B chain of the polynucleotide are separated from the culture medium. The resulting methionines with the A chain and B chain are then removed using cyanogen bromide. The two chains are then chemically purified.
6. Preparation of Insulin Molecule: In sulfonating process, A chain and B chain of nucleotides are combined in presence of sodium disulfonating and sodium sulfite. The two chains are then recombined by a disulfide bond.
7. Purification of insulin: The insulin produced contains the bacteria’s own protein. Therefore the extracted insulin is chemically purified.
8. Insulin Market Types: Pure insulin is filled in suitable ampoules. Then it is marketed.