Open AccessCoupling site‐directed mutagenesis with high‐level expression: large scale production of mutant porins from E. coli
Author(s) -
Prilipov Alexej,
Phale Prashant S,
Gelder Patrick,
Rosenbusch Jurg P,
Koebnik Ralf
Publication year - 1998
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1998.tb13027.x
Subject(s) - porin , mutant , mutagenesis , site directed mutagenesis , biology , bacterial outer membrane , escherichia coli , microbiology and biotechnology , chemistry , biochemistry , gene
Abstract Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli B E host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.