title: Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli.
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 BE 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.
authors.affiliation: Department of Microbiology, Biozentrum, University of Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Prilipov
authors.forename: P S
authors.initials: PS
authors.lastname: Phale
authors.forename: P
authors.initials: P
authors.lastname: Van Gelder
authors.forename: J P
authors.initials: JP
authors.lastname: Rosenbusch
authors.forename: R
authors.initials: R
authors.lastname: Koebnik
mesh_terms: D000251:Adenosine Triphosphatases; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004260:DNA Repair; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018272:Porins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011991:Receptors, Virus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014764:Viral Proteins
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C109237:OmpF protein; D018272:Porins; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C016532:maltoporins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli