Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
2012 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, no 4, 648-659 p.Article in journal (Refereed) Published
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 17 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 17 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 17 RNAP by the 17 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.
Place, publisher, year, edition, pages
2012. Vol. 423, no 4, 648-659 p.
membrane protein production, optimization of protein expression, membrane protein biogenesis, 17 RNA polymerase-based overexpression, membrane protein functional/structural studies
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-106119DOI: 10.1016/j.jmb.2012.07.019ISI: 000310415800017ScopusID: 2-s2.0-84867268590OAI: oai:DiVA.org:kth-106119DiVA: diva2:573607
QC 201212032012-12-032012-11-292012-12-03Bibliographically approved