title: Tuning Escherichia coli for membrane protein overexpression.
abstract: A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Samuel
authors.initials: S
authors.lastname: Wagner
authors.forename: Mirjam M
authors.initials: MM
authors.lastname: Klepsch
authors.forename: Susan
authors.initials: S
authors.lastname: Schlegel
authors.forename: Ansgar
authors.initials: A
authors.lastname: Appel
authors.forename: Roger
authors.initials: R
authors.lastname: Draheim
authors.forename: Michael
authors.initials: M
authors.lastname: Tarry
authors.forename: Martin
authors.initials: M
authors.lastname: Högbom
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: van Wijk
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Jan O
authors.initials: JO
authors.lastname: Persson
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D050576:Membrane Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D020543:Proteome / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D050576:Membrane Fusion Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020543:Proteome; D014764:Viral Proteins; C412443:YIDC protein, E coli; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
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