CLONING AND GENETIC ENGINEERING OF INSULIN
Insulin was the first genetically engineered hormone to be made commercially available for human use. Before cloned human insulin was available, people with diabetes had to give themselves injections of insulin extracted from the pancreas of animals such as cows or pigs. Although this worked well on the whole, occasional allergic reactions occurred, usually to low-level contaminants in the extracts. Today, genuine human insulin (Humulin, marketed by Eli Lilly Inc.) made by recombinant bacteria is available.
If the insulin gene is cloned and directly expressed in bacteria, preproinsulin would be made. Because bacteria lack the mammalian processing enzymes, the preproinsulin would not be converted into insulin (see Fig. 19.15 ). In practice, there are two possible solutions to this problem. The first is to purify the preproinsulin and then treat it with enzymes that convert it into insulin. This means the processing enzymes must be manufactured as well. Clearly this is overly complex. The solution chosen was to make two artificial mini-genes , one for the insulin A-chain and the other for the insulin B-chain ( Fig. 19.18 ).
Two pieces of DNA, encoding the two insulin chains, were synthesized chemically. The two DNA molecules were inserted into plasmids that were put into two separate bacterial hosts. Thus, the two chains of insulin were produced separately by two bacterial cultures. They were then mixed and treated chemically to generate the disulfide bonds linking the chains together.
The approach just described gives insulin that works well. Nonetheless, natural insulin, even natural human insulin, is not perfect, and we can improve on nature. The problem is that natural insulin tends to form hexamers. This clumping covers up the surfaces by which the insulin molecule binds to the insulin receptor, thus preventing most of the insulin from activating its target cells ( Fig. 19.19 ). In vivo, insulin is secreted from the pancreas as a monomer and is distributed rapidly by the bloodstream before it gets a chance to clump. However, when insulin is injected, a high concentration of insulin is present in the syringe and clumping occurs. After injection, it takes a while for the hexamers to dissociate, and it may take several hours for the patient’s blood glucose to drop to normal levels.
Insulin can be genetically engineered to prevent clumping. The DNA sequence of the insulin gene is altered to change the amino acid sequence of the resulting protein. A proline located at the surface where the insulin molecules touch each other when forming the hexamer is replaced with aspartic acid, whose side chain carries a negative charge. So when twomodified insulin molecules approach each other, they are mutually repelled by their negative charges and no longer clump ( Fig. 19.20 ). The altered insulin causes a faster drop in blood sugar than native insulin. In 1999 the Danish pharmaceutical company Novo received approval from the European Union, and the improved insulin may eventually replace the natural product.
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