Physiological Response to Membrane Protein Overexpression in E. coli
2011; Elsevier BV; Volume: 10; Issue: 10 Linguagem: Inglês
10.1074/mcp.m111.007930
ISSN1535-9484
AutoresFrancesca Gubellini, Grégory Verdon, Nathan K. Karpowich, Jon D. Luff, Grégory Boël, Nils C. Gauthier, Samuel K. Handelman, Sarah E. Ades, J.F. Hunt,
Tópico(s)Protein Structure and Dynamics
ResumoOverexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization. Overexpression represents a principal bottleneck in structural and functional studies of integral membrane proteins (IMPs). Although E. coli remains the leading organism for convenient and economical protein overexpression, many IMPs exhibit toxicity on induction in this host and give low yields of properly folded protein. Different mechanisms related to membrane biogenesis and IMP folding have been proposed to contribute to these problems, but there is limited understanding of the physical and physiological constraints on IMP overexpression and folding in vivo. Therefore, we used a variety of genetic, genomic, and microscopy techniques to characterize the physiological responses of Escherichia coli MG1655 cells to overexpression of a set of soluble proteins and IMPs, including constructs exhibiting different levels of toxicity and producing different levels of properly folded versus misfolded product on induction. Genetic marker studies coupled with transcriptomic results indicate only minor perturbations in many of the physiological systems implicated in previous studies of IMP biogenesis. Overexpression of either IMPs or soluble proteins tends to block execution of the standard stationary-phase transcriptional program, although these effects are consistently stronger for the IMPs included in our study. However, these perturbations are not an impediment to successful protein overexpression. We present evidence that, at least for the target proteins included in our study, there is no inherent obstacle to IMP overexpression in E. coli at moderate levels suitable for structural studies and that the biochemical and conformational properties of the proteins themselves are the major obstacles to success. Toxicity associated with target protein activity produces selective pressure leading to preferential growth of cells harboring expression-reducing and inactivating mutations, which can produce chemical heterogeneity in the target protein population, potentially contributing to the difficulties encountered in IMP crystallization. Structural studies of integral membrane proteins (IMPs) 1The abbreviations used are:IMPintegral membrane proteinSRPsignal recognition particleEMelectron microscopyLBLuria brothIPTGisopropyl β-d-thiogalactosideTMtransmembraneABCATP-binding cassetteNBDnucleotide-binding domainIBsinclusion bodiesDICdifferential interference contrastTFtranscription factor. are impeded by many factors, including difficulties associated with their overexpression in simple model organisms like Escherichia coli (1Gordon E. Horsefield R. Swarts H.G. de Pont J.J. Neutze R. Snijder A. Effective high-throughput overproduction of membrane proteins in Escherichia coli.Protein Expr. Purif. 2008; 62: 1-8Crossref PubMed Scopus (54) Google Scholar, 2Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Consequences of membrane protein overexpression in Escherichia coli.Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 3Wagner S. Klepsch M.M. Schlegel S. Appel A. Draheim R. Tarry M. Högbom M. van Wijk K.J. Slotboom D.J. Persson J.O. de Gier J.W. Tuning Escherichia coli for membrane protein overexpression.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 14371-14376Crossref PubMed Scopus (328) Google Scholar, 4Wang D.N. Safferling M. Lemieux M.J. Griffith H. Chen Y. Li X.D. Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies.Biochim. Biophys. Acta. 2003; 1610: 23-36Crossref PubMed Scopus (75) Google Scholar). Toxicity during overexpression often reduces cell growth-rate after induction, contributing to low yield of the target IMP. Studies using many different approaches have investigated the physiology of IMP expression (5White S.H. 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Large-scale production of functional membrane proteins.Cell Mol. Life Sci. 2008; 65: 1729-1755Crossref PubMed Scopus (122) Google Scholar, 10Schlegel S. Klepsch M. Gialama D. Wickström D. Slotboom D.J. de Gier J.W. Revolutionizing membrane protein overexpression in bacteria.Microb. Biotechnol. 2010; 3: 403-411Crossref PubMed Scopus (49) Google Scholar) in E. coli. IMP insertion into the cytoplasmic membrane is believed to be coordinated by carefully regulated interactions between translating ribosomes, the bacterial signal recognition particle (SRP) system (i.e. the Ffh and FtsY proteins and 4.5S RNA), and the SecYEG translocon (5White S.H. Biophysical dissection of membrane proteins.Nature. 2009; 459: 344-346Crossref PubMed Scopus (218) Google Scholar, 6White S.H. von Heijne G. How translocons select transmembrane helices.Annu. Rev. Biophys. 2008; 37: 23-42Crossref PubMed Scopus (162) Google Scholar, 7von Heijne G. Introduction to theme "membrane protein folding and insertion".Annu. 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Membrane protein expression: no cells required.Trends Biotechnol. 2009; 27: 455-460Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Nonetheless, the toxicity frequently observed on IMP overexpression has been attributed to difficulties in accommodating additional IMPs in cellular membranes because of limitations in the capacity of both the enzymes mediating phospholipid biosynthesis and the apparatus mediating IMP insertion (14Drew D. Fröderberg L. Baars L. de Gier J.W. Assembly and overexpression of membrane proteins in Escherichia coli.Biochim. Biophys. Acta. 2003; 1610: 3-10Crossref PubMed Scopus (103) Google Scholar). Destabilization of membranes because of these limitations has been inferred to cause stress impairing the function of membrane-bound enzymes, especially those involved in aerobic respiration (2Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Consequences of membrane protein overexpression in Escherichia coli.Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). integral membrane protein signal recognition particle electron microscopy Luria broth isopropyl β-d-thiogalactoside transmembrane ATP-binding cassette nucleotide-binding domain inclusion bodies differential interference contrast transcription factor. Methods for obtaining a high yield of a native IMP remain primarily empirical and focus on variations in the sequence of the target protein and changes in the expression host. Whole-genome sequence data have been exploited to identify a wide variety of homologous target proteins for evaluation of their expression and stability properties. Variations in affinity-tag and leader-peptide sequences and fusion to expression-enhancing or solubility-enhancing protein domains have yielded improved results for some specific proteins. Published papers have reviewed these approaches, as well as approaches involving variations in growth medium and the use of different E. coli strains or alternative organisms as expression hosts (1Gordon E. Horsefield R. Swarts H.G. de Pont J.J. Neutze R. Snijder A. Effective high-throughput overproduction of membrane proteins in Escherichia coli.Protein Expr. Purif. 2008; 62: 1-8Crossref PubMed Scopus (54) Google Scholar, 4Wang D.N. Safferling M. Lemieux M.J. Griffith H. Chen Y. Li X.D. Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies.Biochim. Biophys. Acta. 2003; 1610: 23-36Crossref PubMed Scopus (75) Google Scholar, 9Junge F. Schneider B. Reckel S. Schwarz D. Dötsch V. Bernhard F. Large-scale production of functional membrane proteins.Cell Mol. Life Sci. 2008; 65: 1729-1755Crossref PubMed Scopus (122) Google Scholar, 10Schlegel S. Klepsch M. Gialama D. Wickström D. Slotboom D.J. de Gier J.W. Revolutionizing membrane protein overexpression in bacteria.Microb. Biotechnol. 2010; 3: 403-411Crossref PubMed Scopus (49) Google Scholar, 15Geertsma E.R. Poolman B. Production of membrane proteins in Escherichia coli and Lactococcus lactis.Methods Mol. Biol. 2010; 601: 17-38Crossref PubMed Scopus (14) Google Scholar, 16Laage R. Langosch D. Strategies for prokaryotic expression of eukaryotic membrane proteins.Traffic. 2001; 2: 99-104Crossref PubMed Scopus (45) Google Scholar). The E. coli strains C41λ(DE3) and C43λ(DE3) (17Miroux B. Walker J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1577) Google Scholar) have been demonstrated to increase the yield of some IMPs as well as some soluble proteins. These strains were selected from the standard BL21λ(DE3) expression host based on their enhanced resistance to the toxicity caused by high-level expression of a specific IMP, the b subunit of the E. coli F1Fo ATPase. Induction of b-subunit expression in these strains causes proliferation of the cytoplasmic membrane (18Arechaga I. Miroux B. Karrasch S. Huijbregts R. de Kruijff B. Runswick M.J. Walker J.E. Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.FEBS Lett. 2000; 482: 215-219Crossref PubMed Scopus (125) Google Scholar), which produces spiral membrane invaginations into the cytoplasm that are visible using thin-section electron microscopy (EM). This phenomenon was also reported after overexpression of the fumarate reductase IMP complex (19Weiner J.H. Lemire B.D. Elmes M.L. Bradley R.D. Scraba D.G. Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle.J. Bacteriol. 1984; 158: 590-596Crossref PubMed Google Scholar) or glycerol-3-phosphate acyltransferase (20Wilkison W.O. Bell R.M. Taylor K.A. Costello M.J. Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.J. Bacteriol. 1992; 174: 6608-6616Crossref PubMed Google Scholar) in standard E. coli strains, but it has yet to be documented for any IMP other than the b-subunit in C41(DE3) or C43(DE3). Moreover, recent genetic analyses have demonstrated that the enhanced yield of IMPs in these strains is attributable primarily to a promoter mutation reducing transcription of T7 RNA polymerase, which lowers its expression and that of target proteins expressed from T7-polymerase-controlled pET vectors. Such vectors were used in the selection procedure that yielded these strains and the subsequently published physiological analyses. Therefore, their main benefit appears to be attenuated expression of the target protein, which can produce higher net yield when expression of that protein is toxic and inhibits cell growth. Other E. coli strains have been selected to improve expression of specific target proteins (21Chen Y. Song J. Sui S.F. Wang D.N. DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli.Protein Expr. Purif. 2003; 32: 221-231Crossref PubMed Scopus (68) Google Scholar, 22Link A.J. Skretas G. Strauch E.M. Chari N.S. Georgiou G. Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.Protein Sci. 2008; 17: 1857-1863Crossref PubMed Scopus (53) Google Scholar, 23Massey-Gendel E. Zhao A. Boulting G. Kim H.Y. 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Other authors have taken a global approach to characterizing the response of E. coli to overexpression of soluble proteins (26Gill R.T. Valdes J.J. Bentley W.E. A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.Metab. Eng. 2000; 2: 178-189Crossref PubMed Scopus (102) Google Scholar, 27Smith H.E. The transcriptional response of Escherichia coli to recombinant protein insolubility.J. Struct. Funct. Genomics. 2007; 8: 27-35Crossref PubMed Scopus (28) Google Scholar) or IMPs (2Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Consequences of membrane protein overexpression in Escherichia coli.Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Gill et al. (26Gill R.T. Valdes J.J. Bentley W.E. A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.Metab. Eng. 2000; 2: 178-189Crossref PubMed Scopus (102) Google Scholar) reported that cells overexpressing soluble proteins from different phylogenetic sources can activate many stress regulons, but noted that the effects of overexpression on cellular growth rate were protein-specific. A more recent analysis employed a proteomics approach to evaluate the response to overexpression of an IMP with a sizable periplasmic domain (2Wagner S. Baars L. Ytterberg A.J. Klussmeier A. Wagner C.S. Nord O. Nygren P.A. van Wijk K.J. de Gier J.W. Consequences of membrane protein overexpression in Escherichia coli.Mol. Cell Proteomics. 2007; 6: 1527-1550Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). These authors propose that the Sec translocon becomes saturated during IMP overexpression, causing accumulation of cytoplasmic aggregates and broad perturbations in the proteome. Some of these perturbations are consistent with inhibition of energy metabolism and cell growth rate due to inefficient oxidative respiration and ATP synthesis in the cytoplasmic membrane. However, these physiological inferences were not evaluated using other methods. Although these published analyses have provided highly valuable data, many issues remain unresolved concerning the physiology of IMP overexpression in E. coli. Practical experience indicates that a substantial number of membrane proteins can be produced in E. coli at suitable levels for structural studies (1–3 mg per liter of culture) without causing toxicity on induction, whereas others are highly toxic even when expressed at undetectable levels. Therefore, uncertainty remains concerning the generality of the phenomena reported in previous literature on IMP overexpression. Therefore, we undertook a systematic analysis of E. coli cells during attempted overexpression of eight target proteins with different expression, toxicity, and folding properties. Two of these were water-soluble cytoplasmic proteins, whereas six were representative polytopic IMPs lacking periplasmic domains. Like most bacterial IMPs without periplasmic domains, these proteins do not have cleavable signal peptides (supplemental Fig. S1). After target-protein induction, we monitored growth rate, morphology, protein expression level, activity of key transcriptional regulators, and global transcriptional profile. Although we identify a large set of physiological changes that occur during IMP overexpression, many of them are shared by cells overexpressing soluble proteins and reflect a likely blockage of the stationary-phase transcriptional program because of some previously unappreciated form of translational stress. We also present evidence that the toxicity caused by overexpression of several IMPs is associated with the biochemical and biophysical properties of these specific proteins. Although definitive conclusions are not possible based on characterization of a relatively restricted set of IMPs, the totality of our results suggests that there may be no intrinsic barrier to moderate overexpression of IMPs in E. coli and that most problems may be attributable to the target protein itself. Target proteins were cloned under the control of the IPTG-inducible T5 promoter in pQE-30, pQE-60, or pQE-70 plasmids (Qiagen Inc., Valencia, CA), which were transformed into strains also containing the LacI-expressing pREP4 accessory plasmid. Unless indicated otherwise, constructs retain the native N termini of the target proteins. EcMsbA*, also called EcMsbA-ΔN5, carries an in-frame deletion of residues 2–5 (HNDK). EcYojI** has a nonsense mutation that truncates the protein after residue 492, deleting half of the central β-sheet in its C-terminal nucleotide-binding domain. HP1206* has six missense mutations that arose during cloning: N65S, M342I, S348T, E363K, D383N, and K432E. EcGlpT has a single Ser inserted after the initiator Met in the native sequence. EcEnolase* contains the catalytically inactivating K341A mutation (28Boël G. Pichereau V. Mijakovic I. Mazé A. Poncet S. Gillet S. Giard J.C. Hartke A. Auffray Y. Deutscher J. Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export?.J. Mol. Biol. 2004; 337: 485-496Crossref PubMed Scopus (59) Google Scholar). NBD-EcMsbA contains the 245 C-terminal residues comprising the cytoplasmic nucleotide-binding domain of EcMsbA. Wild-type EcMsbA was cloned into the pBAD-Myc-His-A vector, and PCR mutagenesis was used to generate the ΔN5 and Nt-His6 variants. The primers used for cloning, sequencing, and mutagenesis are listed in supplemental Table S1. Except as noted below, strains were obtained from the Yale E. coli Genetic Stock Center. Expression experiments were performed in strain MG1655 (F-, λ−, rph-1) transformed with a pQE-derived protein-expression plasmid and pREP4. For reporter-gene assays, these plasmids were transformed into strain SEA001 (MG1655 ΔlacX74, Φλ[rpoHp3::lacZ]) (29Costanzo A. Ades S.E. Growth phase-dependent regulation of the extracytoplasmic stress factor, sigmaE, by guanosine 3′,5′-bispyrophosphate (ppGpp).J. Bacteriol. 2006; 188: 4627-4634Crossref PubMed Scopus (91) Google Scholar) for σE, strain SEA3122 (MG1655 ΔlacX74, λRS88[cpxP-lacZ]) for CpxR, or strain SEA3084 (MG1655 ΔlacX74, Φλ[htpG P1::lacZ]) for σH. Strain glpR-1 (MG1655 glpR-ΔC150) carries a mutation causing the repressor of the glycerol-degradation regulon to be truncated after residue 44. Strain FB20526 (MG1655 fliA::Tn5KAN-2) was obtained from the University of Wisconsin E. coli Genome Project and shown by DNA sequencing to harbor a cryptic glpR-ΔC150 mutation. Strain W3110A (F-, λ−, IN(rrnD-rrnE)1, rph-1) and its derivative WDS2 carrying a temperature-sensitive msbA mutation (30Doerrler W.T. Reedy M.C. Raetz C.R. An Escherichia coli mutant defective in lipid export.J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) were obtained from William T. Doerrler. Cells grown aerobically in Luria broth (LB) at 37 °C were induced at OD600 0.6–0.8 with 1 mM isopropyl β-d-thiogalactoside (IPTG) for 3 h. Cells with pBAD vectors were grown in 0.5% (w/v) glucose before dilution into inducing medium. Small-scale expression experiments employed 8 ml of culture. Membrane solubilization was evaluated using 2% (w/v) βDDM, LDAO, or FC12. See Supplemental Experimental Procedures for details. Reporter-gene activation was monitored using standard methods (29Costanzo A. Ades S.E. Growth phase-dependent regulation of the extracytoplasmic stress factor, sigmaE, by guanosine 3′,5′-bispyrophosphate (ppGpp).J. Bacteriol. 2006; 188: 4627-4634Crossref PubMed Scopus (91) Google Scholar, 31Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, NY1972Google Scholar) to assay the activity of a β-galactosidase fusion carried on a lysogenic λ bacteriophage integrated at a single site on the E. coli chromosome. The σE and CpxR assays employed 0.5 ml samples, while the σH (σ32) assays employed 0.2 ml samples to avoid saturation of OD420 readings. Cells were fixed with 3.7% formaldehyde, mounted on polylysine-coated coverslips, and visualized using a Photometrix CoolSNAP camera after staining with the fluorescent dyes Mitotracker Green or FM4–64 (Invitrogen, Carlsbad, CA). Cells were thin-sectioned as described (18Arechaga I. Miroux B. Karrasch S. Huijbregts R. de Kruijff B. Runswick M.J. Walker J.E. Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.FEBS Lett. 2000; 482: 215-219Crossref PubMed Scopus (125) Google Scholar), stained with uranyl acetate and lead citrate, and imaged using a Philips CM120 transmission electron microscope (FEI, Eindhoven, The Netherlands). Flagella were visualized using a Jeol 100 CX transmission electron microscope (Jeol Ltd., Tokyo, Japan) after staining with 2% (w/v) uranyl acetate. See Supplemental Experimental Procedures for details. RNA extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) 3 h after induction of protein expression was used to synthesize biotinylated cDNA, which was hybridized on Affymetrix E. coli 2.0 arrays by the Gene Expression Center at the University of Wisconsin Biotechnology Center. Raw data (.cel) files were analyzed using the RMA (Robust Multi-chip Average) algorithm in the Affymetrix Expression Console. The transcription-factor-finder software at www.prfect.org used a Fisher's Exact Test with a linear threefold threshold. See Supplemental Experimental Procedures for details. To investigate the effects of IMP overexpression on E. coli physiology, we induced the expression of six inner membrane proteins and two soluble proteins in strain MG1655 (Table I). We selected polytopic IMPs without large periplasmic domains to focus on factors involved in membrane biogenesis and insertion of transmembrane (TM) α-helices. This experimental design isolated factors involved in membrane biogenesis from those involved in secretion of periplasmic protein domains. Typical of bacterial IMPs lacking periplasmic domains, our target proteins do not possess cleavable N-terminal signal peptides based on either predictive algorithms (supplemental Fig. S1) or experimental observations (32Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli.Science. 2003; 301: 616-620Crossref PubMed Scopus (853) Google Scholar, 33Ward A. Reyes C.L. Yu J. Roth C.B. Chang G. Flexibility in the ABC transporter MsbA: Alternating access with a twist.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 19005-19010Crossref PubMed Scopus (648) Google Scholar). We chose two structurally unrelated E. coli IMPs with known crystal structures, GlpT (EcGlpT) and MsbA (EcMsbA*), the Salmonella typhimurium ortholog of MsbA (StMsbA), plus two functionally uncharacterized homologs of MsbA, E. coli YojI (EcYojI), and Helicobacter pylori HP1206*.Table ISummary of protein constructs and expression resultsProteinaThe asterisks indicate proteins harboring mutations (a 5-residue N-terminal truncation for EcMsbA*, a 55-residue C-terminal truncation for EcYojI**, seven missense mutations for HP1206*, and a single missense mutation blocking catalytic activity for EcEnolase*.SpeciesLengthLocationbIM stands for inner membrane.His-tagcAll proteins except EcYojI** were engineered to have hexa-histidine tags at either their amino (N) or carboxy (C) termini; tag sequences are not included in the length indicated in the third column.mRNA InductiondFold-change results from microarray analyses are reported for all replicates for each target protein. Transcripts for NBD-EcMsbA and for proteins from organisms other than E. coli were not detected (n.d.), as explained in the Results section.Protein ExpressionSolubility/Ni-NTA yieldeYields were estimated as all (A), most (M), roughly half (H), or slight (S) based on visual inspection of Coomasie-stained SDS-PAGE gels (supplemental Fig. S1). Extraction was performed without detergent for the soluble proteins or in the presence of the indicated detergents for the membrane proteins. Detergent extracts underwent to microscale batch Ni-NTA purification, except for YojI** because it does not have a his-tag.ToxicityβDDMLDAOFC12EcMsbA*E. coli582IMC12×, 12×++M/MS/MA/MMediumStMsbAS. typhimurium582IMCn.d.++MediumEcYojIE. coli547IMC32×+++S/SS/SM/SNon-toxicEcYojI**E. coli493IM-31×, 37×+++S/n.d.S/n.d.M/n.d.Non-toxicHP1206*H. pylori578IMCn.d.-ToxicEcGlpTE. coli452IMC31×+AfThese estimate are less accurate than the others because of low expression and overlap with other proteins on the SDS-PAGE gel./MMfThese estimate are less accurate than the others because of low expression and overlap with other proteins on the SDS-PAGE gel./MMfThese estimate are less accurate than the others because of low expression and overlap with other proteins on the SDS-PAGE gel./MToxicNBD-EcMsbAE. coli245CytosolCn.d.++H/n.d.Non-toxicEcEnolase*E. coli432CytosolN4×, 4×+++M/n.d.Non-toxica The asterisks indicate proteins harboring mutations (a 5-residue N-terminal truncation for EcMsbA*, a 55-residue C-terminal truncation for EcYojI**, seven missense mutations for HP1206*, and a single missense mutation blocking catalytic activity for EcEnolase*.b IM stands for inner membrane.c All proteins except EcYojI** were engineered to have hexa-histidine tags at either their amino (N) or carboxy (C) termini; tag sequences are not included in the length indicated in the third column.d Fold-change results from microarray analyses are reported for all replicates for each target protein. Transcripts for NBD-EcMsbA and for proteins from organisms other than E. coli were not detected (n.d.), as explained in the
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