DEVELOPMENT OF AN IN-VIVO METHOD TO IDENTIFY MUTANTS OF PHAGE T4-LYSOZYME OF ENHANCED THERMOSTABILITY

An M13 bacteriophage-based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This s ystem takes advantage of easy mutagenesis in an M13 host, the producti on of functional T4 lysozyme during M 1 3 growth, and the ability to d etect lysozyme activity on agar plates. Of several mutagenesis procedu res that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one-step mutagenes is and screening system has been used to find 18 random single-site mu tant lysozymes, of which 11 were heat resistant. Each of these had a m elting temperature within 0.8-1.4-degrees-C of wild type, suggesting t hat the screening system is quite sensitive.