Robustness of hen lysozyme monitored by random mutations
Abstract
Abstract We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg 2+ and Mn 2+ on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.