Overexpression of outer membrane porins in E. coli using pBluescript-derived vectors.
The genes coding for four major outer membrane porins of Escherichia coli, ompF, ompC, phoE, and lamB, have been cloned into pBluescript-derived vectors and overexpressed to very high level (approximately 80% of the total membrane protein) in widely used host strains lacking one or more porins. For OmpF, OmpC, and PhoE porins it is shown that, contrary to current dogma, the genes can be overexpressed without undue deleterious effects upon cell growth and are stable, even under conditions of continuous expression. In contrast, overexpression of LamB is toxic to cell growth, but can be performed using tightly regulated lac promotor-driven expression. The vectors described allow overexpression, sequencing, and mutagenesis to be performed using a single system, without the necessity of subcloning, thus simplifying genetic manipulation. A particular advantage of these new vectors (with the exception of the vector for LamB) is that they do not require a particular regime for inducing the recombinant protein. To our knowledge, this study is the only comparative study of widely used membrane porin expression systems and the first to show that several porins can be stably expressed individually and maintained on high copy number vectors.
Ghosh
R
R
Department of Microbiology, Biozentrum of the University of Basel, Switzerland. Robin.Ghosh@po.uni-stuttgart.de
Steiert
M
M
Hardmeyer
A
A
Wang
Y F
YF
Rosenbusch
J P
JP
D001425:Bacterial Outer Membrane Proteins; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D018272:Porins / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins; D011991:Receptors, Virus; D011994:Recombinant Proteins; C016532:maltoporins; C082922:PhoE protein, E coli