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Tuning Escherichia Coli for Membrane Protein Overexpression

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Tuning Escherichia Coli for Membrane Protein Overexpression Tuning Escherichia coli for membrane protein overexpression Samuel Wagner*†‡, Mirjam M. Klepsch*, Susan Schlegel*, Ansgar Appel*, Roger Draheim*, Michael Tarry*, Martin Ho¨ gbom*, Klaas J. van Wijk§, Dirk J. Slotboom¶, Jan O. Perssonʈ, and Jan-Willem de Gier*†** *Center for Biomembrane Research, Department of Biochemistry and Biophysics, ʈDepartment of Mathematics and Statistics, and †Xbrane Bioscience AB, Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden; §Department of Plant Biology, Cornell University, Ithaca, NY 14853; and ¶Department of Biochemistry, University of Groningen, Nyenborg 4, 9747 AG Groningen, The Netherlands Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved July 30, 2008 (received for review April 28, 2008) A simple generic method for optimizing membrane protein over- produce low amounts of T7Lys, whereas pLysE hosts produce expression in Escherichia coli is still lacking. We have studied the much more enzyme and, therefore, provide a more stringent physiological response of the widely used ‘‘Walker strains’’ control (6). C41(DE3) and C43(DE3), which are derived from BL21(DE3), to Recently, we studied the physiological response of E. coli membrane protein overexpression. For unknown reasons, overex- BL21(DE3)pLysS to membrane protein overexpression (7). Our pression of many membrane proteins in these strains is hardly aim was to identify potential bottlenecks that hamper membrane toxic, often resulting in high overexpression yields. By using a protein overexpression and to use this information to engineer combination of physiological, proteomic, and genetic techniques strains with improved overexpression characteristics. We found we have shown that mutations in the lacUV5 promoter governing that membrane protein overexpression resulted in accumulation expression of T7 RNA polymerase are key to the improved mem- of cytoplasmic aggregates containing the overexpressed protein brane protein overexpression characteristics of the Walker strains. as well as chaperones, proteases, many essential cytoplasmic Based on this observation, we have engineered a derivative strain proteins, and many precursors of periplasmic and outer mem- of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of brane proteins. Also, levels of respiratory chain complexes in the the T7 RNA polymerase can be precisely controlled by its natural cytoplasmic membrane were strongly reduced, causing the in- inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for mem- duction of the AcO-pta pathway for ATP production and brane protein overexpression and conveniently allows optimizing downregulation of the tricarboxylic acid (TCA) cycle, resulting overexpression of any given membrane protein by using only a in inefficient ATP production. Presumably, these effects were single strain rather than a multitude of different strains. The caused by saturation of the Sec translocon, which mediates both generality and simplicity of our approach make it ideal for high- translocation of secretory proteins across and integration of throughput applications. membrane proteins into the cytoplasmic membrane. As a complementary approach, we decided to characterize the engineering ͉ systems biotechnology ͉ proteomics so-called Walker strains C41(DE3) and C43(DE3). These strains were selected almost a decade ago in a screen that was designed he natural abundance of membrane proteins is typically too to isolate derivatives of BL21(DE3) with improved membrane Tlow to isolate sufficient amounts of material for functional protein overexpression characteristics (8). Overexpression of and structural studies. Therefore, membrane proteins must be many membrane proteins in these strains is hardly toxic, often obtained by overexpression, and the bacterium E. coli is the most resulting in high overexpression yields (8). The reason for their widely used vehicle for this purpose (1). Although many mem- improved membrane protein overexpression characteristics is brane proteins can be overexpressed in inclusion bodies, their not understood. refolding into functional proteins is often not successful (2). To Here, we report an in-depth characterization of the Walker avoid the refolding problem, overexpression of membrane pro- strains by using a combination of 1D- and 2D-gel electrophoresis teins by accumulation in the cytoplasmic membrane is needed. and mass spectrometry, complemented with Western blotting, However, overexpression is often toxic to the cell, thereby enzymatic activity assays, flow cytometry, and genetics. This preventing biomass formation and severely reducing yields (1). analysis has allowed us to identify the key mutations behind the Thus, membrane protein overexpression has to be optimized, but improved membrane overexpression characteristics. Based on no systematic, generic, and high-throughput-compatible method this previously undescribed insight, we have engineered an E. coli is available for the optimization process. strain that is tunable for membrane protein overexpression and Bacteriophage T7 RNA polymerase (T7RNAP) is often used conveniently allows optimizing overexpression of any given to drive recombinant protein production in E. coli (3). In protein by using only a single strain rather than a multitude of BL21(DE3) and its derivatives, the gene encoding T7RNAP is different strains. under control of the lacUV5 promoter, a strong variant of the wild-type lac promoter. It is insensitive to catabolite repression and, therefore, controlled only by the lac repressor, LacI, which Author contributions: S.W., M.M.K., S.S., D.J.S., J.O.P., and J.-W.d.G. designed research; S.W., M.M.K., S.S., A.A., D.J.S., and J.O.P. performed research; R.D., M.T., and M.H. con- binds to the lac operator (4). T7RNAP exclusively recognizes the tributed new reagents/analytic tools; S.W., M.M.K., S.S., A.A., K.J.v.W., D.J.S., J.O.P., and T7 promoter and it transcribes eight times faster than E. coli J.-W.d.G. analyzed data; and S.W., K.J.v.W., and J.-W.d.G. wrote the paper. BIOCHEMISTRY RNAP allowing high yield protein production (5). Most T7 Conflict of interest statement: S.W. and J.-W.d.G. are cofounders of the biotech company expression vectors employ a T7lac hybrid promoter that com- Xbrane Bioscience AB. bines the strong T7 ␾10 promoter with a lac operator to diminish This article is a PNAS Direct Submission. leaky expression. On addition of the inducer isopropyl ␤-D- ‡Present address: Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, thiogalactoside (IPTG), lacI repression is relieved, resulting in Yale School of Medicine, New Haven, CT 06536. recombinant protein production. If toxicity due to leaky expres- **To whom correspondence should be addressed. E-mail: [email protected]. sion is a problem, T7RNAP activity can be further dampened This article contains supporting information online at www.pnas.org/cgi/content/full/ with the T7RNAP inhibitor T7Lys (6). For T7Lys expression, 0804090105/DCSupplemental. usually the pLysS and pLysE plasmids are used; pLysS hosts © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804090105 PNAS ͉ September 23, 2008 ͉ vol. 105 ͉ no. 38 ͉ 14371–14376 Downloaded by guest on September 24, 2021 A BCD 3.5 14000 BL21pLysS control BL21pLysS C41 3 12000 BL21pLysS YidC-GFP C41 control 2.5 10000 C41 YidC-GFP C43 control YidC-GFP YidC-GFP 2 8000 C43 YidC-GFP 600 A 1.5 6000 1 4000 side scatter side scatter 0.5 2000 control control 0 0 -100 0 100 200 300 fluorescence/GFP ml RFU/ 0 50 100 150 200 250 300 time after induction/ min time after induction/ min forward scatter forward scatter EFG120 C43 BL21pLysS 100 controls C43 C41 80 YidC-GFP 60 40 cell counts cell side scatter BL21pLysS control BL21pLysS YidC-GFP 20 C41 control C41 YidC-GFP control C43 control C43 YidC-GFP relative oxygen consumption 0 1 1.5 2 2.5 3 3.5 4 forward scatter GFP time after induction/ h Fig. 1. Analysis of growth, protein expression, morphology, and respiration of BL21(DE3)pLysS, C41(DE3), and C43(DE3) overexpressing YidC-GFP. (A and B) Growth (A) and protein expression of cells overexpressing YidC-GFP (B) were monitored by measuring the A600 and GFP fluorescence, respectively, every 30 min. (C–F) The following parameters were monitored by flow cytometry: forward and side scatter (C–E), which provide information about cell size and granularity, and GFP fusion protein expression (F). For C–F, cells were harvested 4 h after induction with IPTG. (G) Oxygen consumption was measured in whole cells every hour. Experiments were done in triplicate. Respiratory activities of control cells were set to 100. Results efficient respiration due to a less severe perturbation of the To compare the consequences of membrane protein overexpres- cytoplasmic membrane proteome. Surprisingly, after4hof sion in C41(DE3), C43(DE3), and BL21(DE3)pLysS, we used YidC-GFP overexpression, the cytoplasmic membrane pro- the well characterized YidC-GFP fusion as the initial test protein teomes of C41(DE3) and C43(DE3) were affected in a similar (7). In this fusion, the membrane protein YidC is C-terminally way as in BL21(DE3)pLysS (Figs. S1 and S2A and Table S1). fused to GFP. YidC-GFP is predominantly overexpressed in the This observation was corroborated by enzymatic activity assays membrane and its GFP-moiety allows convenient monitoring of of some key complexes involved in energy transduction (Fig. overexpression levels by using fluorescence detection (9, 10). S2B). However, when oxygen consumption was measured over time it became clear that on induction of YidC-GFP overex- Growth, Overexpression, and Morphology. YidC-GFP overexpres- pression the decrease in oxygen consumption was instantaneous sion affected growth of both C41(DE3) and C43(DE3) much less in BL21(DE3)pLysS, but only gradual in C41(DE3) and than of BL21(DE3)pLysS (Fig. 1A). After 6-h induction, yields C43(DE3) (Fig. 1G). were 4- and 6-fold higher in cultures of C41(DE3) and The accumulation levels of the chaperones ClpB and IbpA, C43(DE3), compared with the BL21(DE3)pLysS culture (Fig.
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