Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability.


Abstract

An M13 bacteriophage-based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates. Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one-step mutagenesis and screening system has been used to find 18 random single-site mutant lysozymes, of which 11 were heat resistant. Each of these had a melting temperature within 0.8-1.4 degrees C of wild type, suggesting that the screening system is quite sensitive. Study holds ProTherm entries: 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 13505, 13506, 13507, 13508, 13509, 13510, 13511, 13512, 13513, 13514, 13515, 13516, 13517 Extra Details: cysteine-free pseudo wild type lysozyme, 1L63 (C54T, C97A) protein stability; screening;mutagenesis

Submission Details

ID: 4MUZChVo

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Pjura P;Matsumura M;Baase WA;Matthews BW,Protein Sci. (1993) Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability. PMID:7507755
Additional Information

Sequence Assay Result Units