title: Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3).
abstract: Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Susan
authors.initials: S
authors.lastname: Schlegel
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.forename: Chiara
authors.initials: C
authors.lastname: Lee
authors.forename: Anna
authors.initials: A
authors.lastname: Hjelm
authors.forename: Mirjam
authors.initials: M
authors.lastname: Klepsch
authors.forename: Marc
authors.initials: M
authors.lastname: Strous
authors.forename: David
authors.initials: D
authors.lastname: Drew
authors.forename: Dirk Jan
authors.initials: DJ
authors.lastname: Slotboom
authors.forename: Jan-Willem
authors.initials: JW
authors.lastname: de Gier
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009238:N-Acetylmuramoyl-L-alanine Amidase