A genetic screen for mutations that increase the thermal stability of phage T4 lysozyme.

Abstract

A method was developed to screen for mutants of phage T4 lysozyme that are more stable than the wild-type enzyme. Using an assay that detects lysozyme activity on Petri plates, protein synthesized during the formation of phage plaques at a permissive temperature (33.degree. C) was tested for is ability to withstand incubation at a temperature that inactivates the wild-type enzyme. In the initial screen of .apprx. 3 .times. 104 plaques from a T4 phage stock mutagenized with hydroxylamine, > 30 mutant that produce lysozyme activity resistant to high temperature incubation were found. Lysozyme produced by 2 of the mutants was purified and found to denature at a higher temperature than the wild-type enzyme in vitro. Such mutants were called st for thermostable. The existence of st mutants indicates that protein stability is not maximized during evolution; instead, it is likely that stability is optimized for the phyisiology of the organism. Analysis of the structures of these mutants will provide another way to identify and predict interactions that stabilize proteins. The method of finding thermostable variants presented here may be applicable to any protein that can be detected by a plate assay or by a plate screen with antibodies.