Premium
Development of an in vivo method to identify mutants of phage T4 lysozyme of enhanced thermostability
Author(s) -
Pjura Philip,
Matsumura Masazumi,
Baase Walter A.,
Matthews Brian W.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560021221
Subject(s) - lysozyme , thermostability , mutant , mutagenesis , bacteriophage , directed evolution , site directed mutagenesis , reverse transcriptase , biology , in vivo , protein engineering , chemistry , enzyme , biochemistry , genetics , polymerase chain reaction , gene , escherichia coli
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°C of wild type, suggesting that the screening system is quite sensitive.