Immunological tests for missense mutants

Temperature sensitive mutants are nearly always due to missense mutations in a structural gene for a protein. With this exception, it is usually difficult to determine if a mutation which causes complete loss of an enzymatic activity is due to a missense mutation or some other type of mutation. One way to identify missense mutations affecting a particular gene is to test for the presence of protein using a polyclonal antibody that specifically recognizes that protein. A positive antibody reaction is described as immunological cross-reacting material or CRM for short. (If you need a brief review of antibodies, see the website Introduction to Antibodies).

Testing for CRM is not trivial. It requires antibody to the wild-type protein. To make polyclonal antibody against a particular protein, the protein must first be purified. The purified protein is then injected into animals (e.g. rabbits) to stimulate the production of antibodies that recognize the protein. A good antibody response typically requires a booster injection also. The process usually takes many weeks before an adequate level of antibodies have been developed. Serum is then collected from the animals and assayed for specific antibodies to the wild-type protein in vitro.

Once the specific antisera has been produced, it can be used to test for the presence of the protein in cell extracts. Even though the protein may be inactive due to a missense mutation, if the protein is made by the mutant the extract will have cross-reacting material (CRM+). However, if the mutant does not make the protein or if the protein is rapidly degraded, then cross-reacting material will not be detected in the extract (CRM-). The tests for CRM can be done in microtiter dishes with 96 wells, allowing many mutants to be tested at one time. Thus, although this process of obtaining the antibody is rather slow, once the specific antibody is available, it is possible to rapidly test for mutants that make the inactive protein using simple immunological tests.

Some questions to ponder:

Monoclonal antibodies can also be used to test for CRM. What is a possible disadvantage of using monoclonal antibodies for this purpose instead of polyclonal antibodies?

Which of the following types of mutants would be likely to produce CRM? In each case, explain why or why not.

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Last modified July 5, 2004