Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3) |
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Authors: | Susan SchlegelJohn Löfblom Chiara LeeAnna Hjelm Mirjam KlepschMarc Strous David DrewDirk Jan Slotboom Jan-Willem de Gier |
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Institution: | 1 Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden2 School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden3 Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK4 Institute for Genome Research and Systems Biology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, D-33615 Bielefeld, Germany5 Max Planck Institute for Marine Microbiology, Celsiusstraße 1, D‐28359 Bremen, Germany6 Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands |
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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. |
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Keywords: | RNAP RNA polymerase lys lysozyme GFP green fluorescent protein LB lysogeny broth IbpB inclusion body binding protein B FACS fluorescence-activated cell sorting SEC size-exclusion chromatography PBS phosphate-buffered saline DDM d-maltoside" target="_blank">n-dodecyl-β-d-maltoside CV column volume |
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