A diagram of the manufacturing steps for insulin.
Insulin contains two short polypeptide chains - chain A and chain B linked by disulphide bridges. In mammals, insulin is synthesized as a pro hormone(that needs to be processed to become mature and functional hormone ).It contains an extra stretch called C- peptide. C-peptide is removed during maturation into insulin, hence it is absent in mature insulin. The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.
Let's see the steps involved in the production of insulin from the tiny bacterium. The control of all the normal activities of a bacterium depends upon its single chromosome and small rings of genes called plasmids. In genetic engineering pieces of chromosome from a different organism can be inserted into a plasmid. This allows the bacteria to make a new substance.
We need to know the steps of this process. They are:
- The gene for producing human insulin protein is isolated. The gene is part of the DNA in a human
- they use specific restriction enzymes to cut the insulin gene out of the chromosome
- plasmids are then removed from bacterial cells (Why plasmids - the main purpose of a plasmid is to serve as a vehicle for gene of interest, we can insert the human insulin gene into the plasmid. This is the purpose of a multiple cloning site. A multiple cloning site is the location in a plasmid where a sequence of DNA, typically a gene, can be inserted.)
- the plasmids are cut open with an enzyme
- a human insulin gene is inserted into each plasmid
- and the plasmids with gene of interest are again incorporated into the bacterium and now the genetic engineers encourage the bacteria to accept the genetically modified plasmids
- bacteria with the insulin gene are then multiplied in the culture medium
- each bacterium will produce a tiny volume of insulin
- by culturing the genetically engineered bacteria, limitless supplies of insulin may be produced. Hence, the newly formed insulin is collected and synthesized for further processing. This process is not only used for producing insulin, but genetic engineering is used for the production of substances which used to be both expensive and difficult to produce. Examples include:
- antibiotics such as penicillin
- various vaccines for the control of disease (For example; hormones, such as the human growth hormone (HGH), can be formulated through recombinant DNA technology; for example, HGH can be cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector.