COUPLING SITE-DIRECTED MUTAGENESIS WITH HIGH-LEVEL EXPRESSION - LARGE-SCALE PRODUCTION OF MUTANT PORINS FROM ESCHERICHIA-COLI

Combination of an origin repair mutagenesis system with a new mutS hos t strain increased the efficiency of mutagenesis from 46% to 75% mutan t clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli B-E ho st strains devoid of major outer membrane proteins was constructed, fa cilitating the purification of mutant porins to homogeneity. This allo wed preparation of 149 porin mutants in E. coli used in detailed explo rations of the structure and function of this membrane protein to high resolution. (C) 1998 Federation of European Microbiological Societies . Published by Elsevier Science B.V. All rights reserved.