Hypersynthesis of Proteins
Two reasons for cloning genes are to mutate the gene to alter the product for in vivo or in vitro studies and to increase the synthesis of a gene product either in its native organism or in bacteria. Hypersynthesis almost always seems necessary, for the more interesting the protein, the lower the levels at which it seems to be synthesized.
The most important factors limiting levels of protein synthesis in bacteria, which is where most cloned genes are expressed, are the strength of the promoter and the ribosome binding site. Secondary factors that can affect translation are the potential for folding of the mRNA and codon usage. One of the most important and most vexing problems in the hypersynthesis of proteins is proper folding. A high synthesis rate of many proteins does not yield a high concentration of the protein in the cytoplasm of the cells. Instead the protein is found in the form of granules or pellets often approaching the size of a bacterial cell. These are called inclusion bodies.
Inclusion bodies form when the protein synthesis rate so greatly exceeds its folding rate that an excess of hydrophobic areas are exposed. These bind and aggregate to form amorphous precipitates of inactive, and often insoluble protein. Several steps sometimes reduce the prob-lem. Cells can be grown at low temperatures so that the rate of protein synthesis is greatly slowed whereas the rate of folding is only slightly depressed. The inclusion bodies can easily be purified by low speed centrifugation. They often contain essentially pure protein and occa-sionally the protein can be dissolved in urea or guanidine hydrochloride and renatured to yield active protein by slow removal of these denatur-ing agents. Sometimes the presence of chaperonin proteins during the synthesis or renaturation process increases yields of soluble protein.