Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies

Pankaj Panwar1, M. Joanne Lemieux1

1 Department of Biochemistry, Membrane Protein Disease Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.9
DOI:  10.1002/0471140864.ps2909s76
Online Posting Date:  April, 2014
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Abstract

Rhomboid proteases are membrane‐embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC). Curr. Protoc. Protein Sci. 76:29.9.1‐29.9.25 © 2014 by John Wiley & Sons, Inc.

Keywords: rhomboid protease; expression study; pBAD; IMAC; SEC

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Optimization of Induction Temperature for Rhomboid Expression
  • Basic Protocol 2: Optimization of Inducing Agent Concentration for Rhomboid Expression
  • Basic Protocol 3: Optimization of Induction Time for Rhomboid Expression
  • Basic Protocol 4: Large‐Scale Expression of Rhomboid Protease and Membrane Fractionation
  • Basic Protocol 5: Large‐Scale Purification of Rhomboid Protease by Ion Metal Affinity Chromatography Followed by Thrombin Cleavage and Size‐Exclusion Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Optimization of Induction Temperature for Rhomboid Expression

  Materials
  • Cloned expression vector pBAD‐MycHis (Invitrogen)
  • TOP10 cells (Invitrogen)
  • LB‐amp plates (see recipe)
  • LB‐amp (see recipe)
  • 2% (w/v) arabinose (Sigma‐Aldrich)
  • PBS (see recipe)
  • 0.5 M EDTA (Sigma‐Aldrich)
  • 100 mM PMSF (Sigma‐Aldrich)
  • 10× PIC (see recipe)
  • 10 mg/ml DNase (Sigma‐Aldrich)
  • 10% (v/v) Triton X‐100 (Sigma‐Aldrich)
  • 2× SDS‐PAGE buffer (see recipe)
  • SDS‐PAGE gel, freshly prepared
  • Mouse anti‐His antibody (Santa Cruz Biotech)
  • 37°C incubator with shaking
  • 250‐ and 500‐ml Erlenmeyer flasks (Fisher Scientific)
  • Spectrophotometer
  • 50‐ml Falcon tubes
  • J26 centrifuge (Beckman)
  • Refrigerated shaker (New Brunswick)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • SDS‐PAGE apparatus (BioRad)
  • Western blot apparatus (BioRad)

Basic Protocol 2: Optimization of Inducing Agent Concentration for Rhomboid Expression

  Materials
  • Cloned expression vector pBAD‐MycHis (Invitrogen)
  • Top10 cells (Invitrogen)
  • LB‐amp agar plates (see recipe)
  • LB‐amp (see recipe)
  • 2% (w/v) arabinose (Sigma‐Aldrich)
  • PBS (see recipe)
  • 0.5 M EDTA (Sigma‐Aldrich)
  • 100 mM PMSF (Sigma‐Aldrich)
  • 10× PIC (see recipe)
  • 10 mg/ml DNase (Sigma‐Aldrich)
  • 10% (v/v) Triton X‐100 (Sigma‐Aldrich)
  • 2× SDS‐PAGE buffer (see recipe)
  • SDS‐PAGE gel
  • PVDF membrane
  • Mouse anti‐His antibody (Santa Cruz Biotech)
  • 37°C incubators with and without shaker
  • 250‐ and 500‐ml Erlenmeyer flasks (Fisher Scientific
  • Spectrophotometer
  • 50‐ml centrifuge tubes (Falcon)
  • J26 centrifuge (Beckman)
  • Refrigerated shaker (New Brunswick)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • SDS‐PAGE apparatus (BioRad)
  • Western blot apparatus (BioRad)

Basic Protocol 3: Optimization of Induction Time for Rhomboid Expression

  Materials
  • Cloned expression vector pBAD‐MycHis (Invitrogen)
  • Top10 cells (Invitrogen)
  • LB‐amp plates (see recipe)
  • LB‐amp (see recipe)
  • 2% (w/v) arabinose (Sigma‐Aldrich)
  • PBS (see recipe)
  • 0.5 M EDTA (see recipe)
  • 100 mM PMSF (see recipe)
  • 10× PIC (see recipe)
  • 10 mg/ml DNase (see recipe)
  • 10% (v/v) Triton X‐100 (see recipe)
  • 2× SDS‐PAGE buffer (see recipe)
  • SDS‐PAGE gel, freshly poured
  • Mouse anti‐His antibody (Santa Cruz Biotech)
  • 37°C incubator with and without shaking
  • 500‐ml and 4‐liter Erlenmeyer flasks (Fisher Scientific)
  • Spectrophotometer
  • 50‐ml tubes (Falcon)
  • J26 centrifuge (Beckman)
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated shaker (New Brunswick)
  • Refrigerated microcentrifuge
  • SDS‐PAGE apparatus (BioRad)
  • Western blot apparatus (BioRad)

Basic Protocol 4: Large‐Scale Expression of Rhomboid Protease and Membrane Fractionation

  Materials
  • Cloned expression vector
  • Top10 cells (Invitrogen)
  • LB‐amp plates (see recipe)
  • LB‐amp (see recipe)
  • 2% (w/v) arabinose (see recipe)
  • Lysis buffer (see recipe)
  • 37°C incubators with and without shaking
  • 4‐liter Erlenmeyer flasks (Fisher Scientific)
  • Spectrophotometer
  • J26 centrifuge (Beckman)
  • EmulsiFlex‐C5 microfluidizer processer (Avestin)
  • Ultracentrifuge Ti‐45 rotor and 65‐ml tubes (Beckman)
  • Optima L‐100k ultracentrifuge (Beckman)
NOTE: All steps are performed at 4°C unless otherwise stated.

Basic Protocol 5: Large‐Scale Purification of Rhomboid Protease by Ion Metal Affinity Chromatography Followed by Thrombin Cleavage and Size‐Exclusion Chromatography

  Materials
  • 4 to 5 g membrane fraction containing 10 to 20 mg of hiGlpG (obtained from 6 liters cell culture in LB from protocol 4)
  • Solubilization buffer (see recipe)
  • Dodecylmaltoside (DDM, Anatrace)
  • IMAC resin: Ni2+‐NTA (Thermo Scientific)
  • IMAC buffer (see recipe)
  • IMAC wash buffer (see recipe)
  • IMAC elution buffer 1‐4 (see recipe)
  • BCA protein assay kit (BioRad), optional
  • SEC buffer (see recipe)
  • Thrombin (GE Healthcare)
  • 50‐ml Wheaton Potter‐Elvehjem tissue grinder (glass homogenizer)
  • Beakers
  • Magnetic stir bar and plate
  • 65‐ml ultracentrifuge tubes
  • Refrigerated ultracentrifuge
  • Rocking shaker (Fisher Scientific)
  • 25‐ml poly prep drip column (BioRad)
  • Spectrophotometer (Fisher Scientific)
  • Superdex‐200 16/60 GL column (GE healthcare)
  • Dialysis tubing, 12‐ to 14‐kDa molecular weight cutoff (MWCO, Spectrum Labs)
  • 30‐kDa MWCO concentrator (Amicon)
  • ÄKTA FPLC (GE Healthcare)
  • Additional reagents and equipment for SDS PAGE (unit )
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Figures

Videos

Literature Cited

Literature Cited
   Alexandrov, A.I. , Mileni, M. , Chien, E.Y. , Hanson, M.A. , and Stevens, R.C. 2008. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16:351‐359.
   Auer, M. , Kim, M.J. , Lemieux, M.J. , Villa, A. , Song, J. , Li, X.D. , and Wang, D.N. 2001. High‐yield expression and functional analysis of Escherichia coli glycerol‐3‐phosphate transporter. Biochemistry 40:6628‐6635.
   Ben‐Shem, A. , Fass, D. , and Bibi, E. 2007. Structural basis for intramembrane proteolysis by rhomboid serine proteases. Proc. Natl. Acad. Sci. U.S.A. 104:462‐466.
   Bill, R.M. , Henderson, P.J. , Iwata, S. , Kunji, E.R. , Michel, H. , Neutze, R. , Newstead, S. , Poolman, B. , Tate, C.G. , and Vogel, H. 2011. Overcoming barriers to membrane protein structure determination. Nat. Biotechnol. 29:335‐340.
   Bradd, S. J. and Dunn, M. J. 1993. Analysis of membrane proteins by western blotting and enhanced chemiluminescence. Methods Mol. Biol. 19:211‐218
   Brooks, C.L. , Lazareno‐Saez, C. , Lamoureux, J.S. , Mak, M.W. , and Lemieux, M.J. 2011. Insights into substrate gating in H. influenzae rhomboid. J. Mol. Biol. 407:687‐697.
   Brooks, C.L. , Morisson, M.A. , and Lemieux, M.J. 2013. Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris . Protein Sci. 22:425‐433.
   Carrio, M.M. and Villaverde, A. 2002. Construction and deconstruction of bacterial inclusion bodies. J. Biotechnol. 96:3‐12.
   Chen, Y. , Song, J. , Sui, S.F. , and Wang, D.N. 2003. DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli . Protein Expr. Purif. 32:221‐231.
   Czyzewski, B.K. and Wang, D.N. 2012. Identification and characterization of a bacterial hydrosulphide ion channel. Nature 483:494‐497.
   DaCosta, C.J.B. and Baenziger, J.E. 2002. A rapid method for assessing lipid: Protein and detergent: Protein ratios in membrane‐protein crystallization. Acta Crystallogr. D Biol. Crystallogr. 59:77‐83.
   Deniaud, A. , Panwar, P. , Frelet‐Barrand, A. , Bernaudat, F. , Juillan‐Binard, C. , Ebel, C. , Rolland, N. , and Pebay‐Peyroula, E. 2012. Oligomeric status and nucleotide binding properties of the plastid ATP/ADP transporter 1: Toward a molecular understanding of the transport mechanism. PloS One 7:e32325.
   Dong, H. , Nilsson, L. , and Kurland, C.G. 1995. Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J. Bacteriol. 177:1497‐1504.
   Drew, D. , Newstead, S. , Sonoda, Y. , Kim, H. , von Heijne, G. , and Iwata, S. 2008. GFP‐based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae . Nat. Protoc. 3:784‐798.
   Freeman, M. 2008. Rhomboid proteases and their biological functions. Annu. Rev. Genet. 42:191‐210.
   Guzman, L.M. , Belin, D. , Carson, M.J. , and Beckwith, J. 1995. Tight regulation, modulation, and high‐level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121‐4130.
   Huang, Y. , Lemieux, M.J. , Song, J. , Auer, M. , and Wang, D.N. 2003. Structure and mechanism of the glycerol‐3‐phosphate transporter from Escherichia coli . Science 301:616‐620.
   Kawate, T. and Gouaux, E. 2006. Fluorescence‐detection size‐exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673‐681.
   Lazareno‐Saez, C. , Arutyunova, E. , Coquelle, N. , and Lemieux, M.J. 2013. Domain swapping in the cytoplasmic domain of the Escherichia coli rhomboid protease. J. Mol. Biol. 425:1127‐1142.
   Lemieux, M.J. , Reithmeier, R.A. , and Wang, D.N. 2002. Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion‐exchanger membrane domain. J. Struct. Biol. 137:322‐332.
   Lemieux, M.J. , Song, J. , Kim, M.J. , Huang, Y. , Villa, A. , Auer, M. , Li, X.D. , and Wang, D.N. 2003. Three‐dimensional crystallization of the Escherichia coli glycerol‐3‐phosphate transporter: A member of the major facilitator superfamily. Protein Sci. 12:2748‐2756.
   Lemieux, M.J. , Fischer, S.J. , Cherney, M.M. , Bateman, K.S. , and James, M.N. 2007. The crystal structure of the rhomboid peptidase from Haemophilus influenzae provides insight into intramembrane proteolysis. Proc. Natl. Acad. Sci. U.S.A. 104:750‐754.
   Lunin, V.V. , Dobrovetsky, E. , Khutoreskaya, G. , Zhang, R. , Joachimiak, A. , Doyle, D.A. , Bochkarev, A. , Maguire, M.E. , Edwards, A.M. , and Koth, C.M. 2006. Crystal structure of the CorA Mg2+ transporter. Nature 440:833‐837.
   Magnusdottir, A. , Johansson, I. , Dahlgren, L.G. , Nordlund, P. , and Berglund, H. 2009. Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates. Nat. Methods 6:477‐478.
   Mancusso, R. , Gregorio, G.G. , Liu, Q. , and Wang, D.N. 2012. Structure and mechanism of a bacterial sodium‐dependent dicarboxylate transporter. Nature 491:622‐626.
   Miroux, B. and Walker, J.E. 1996. 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. 260:289‐298.
   Mohanty, A.K. and Wiener, M.C. 2004. Membrane protein expression and production: Effects of polyhistidine tag length and position. Protein Expr. Purif. 33:311‐325.
   Narayanan, A. , Ridilla, M. , and Yernool, D.A. 2011. Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator‐repressor interactions. Protein Sci. 20:51‐61.
   Oberg, F. , Sjohamn, J. , Conner, M.T. , Bill, R.M. , and Hedfalk, K. 2011. Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: The importance of codon optimization and clone selection. Mol. Membr. Biol. 28:398‐411.
  O' Malley, M.A. , Helgeson, M.E. , Wagner, N.J. , and Robinson, A.S. 2011. The morphology and composition of cholesterol‐rich micellar nanostructures determine transmembrane protein (GPCR) activity. Biophys. J. 100:11‐13.
   Porath, J. , Carlsson, J. , Olsson, I. , and Belfrage, G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598‐599.
   Sahin, E. and Roberts, C.J. 2012. Size‐exclusion chromatography with multi‐angle light scattering for elucidating protein aggregation mechanisms. Methods Mol. Biol. 899:403‐423.
   Sampathkumar, P. , Mak, M.W. , Fischer‐Witholt, S.J. , Guigard, E. , Kay, C.M. , and Lemieux, M.J. 2012. Oligomeric state study of prokaryotic rhomboid proteases. Biochim. Biophys. Acta 1818:3090‐3097.
   Seddon, A.M. , Curnow, P. , and Booth, P.J. 2004. Membrane proteins, lipids and detergents: Not just a soap opera. Biochim. Biophys. Acta 1666:105‐117.
   Serrano‐Vega, M.J. , Magnani, F. , Shibata, Y. , and Tate, C.G. 2008. Conformational thermostabilization of the beta1‐adrenergic receptor in a detergent‐resistant form. Proc. Natl. Acad. Sci. U.S.A. 105:877‐882.
   Sonoda, Y. , Newstead, S. , Hu, N.J. , Alguel, Y. , Nji, E. , Beis, K. , Yashiro, S. , Lee, C. , Leung, J. , Cameron, A.D. , Byrne, B. , Iwata, S. , and Drew, D. 2011. Benchmarking membrane protein detergent stability for improving throughput of high‐resolution X‐ray structures. Structure 19:17‐25.
   Tate, C.G. 2010. Practical considerations of membrane protein instability during purification and crystallisation. Methods Mol. Biol. 601:187‐203.
   Vinothkumar, K.R. , Strisovsky, K. , Andreeva, A. , Christova, Y. , Verhelst, S. , and Freeman, M. 2011. The structural basis for catalysis and substrate specificity of a rhomboid protease. EMBO J. 29:3797‐3809.
   Wagner, S. , Baars, L. , Ytterberg, A.J. , Klussmeier, A. , Wagner, C.S. , Nord, O. , Nygren, P.A. , van Wijk, K.J. , and de Gier, J.W. 2007. Consequences of membrane protein overexpression in Escherichia coli . Mol. Cell. Proteom. 6:1527‐1550.
   Wang, D.N. , Lemieux, M.J. , and Boulter, J.M. 2003. Purification and characterization of transporter proteins from human erythrocyte membrane. Methods Mol. Biol. 228:239‐255.
   Wang, Y. , Zhang, Y. , and Ha, Y. 2006. Crystal structure of a rhomboid family intramembrane protease. Nature 444:179‐180.
   Wiener, M.C. 2004. A pedestrian guide to membrane protein crystallization. Methods 34:364‐372.
   Wu, Z. , Yan, N. , Feng, L. , Oberstein, A. , Yan, H. , Baker, R.P. , Gu, L. , Jeffrey, P.D. , Urban, S. , and Shi, Y. 2006. Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry. Nat. Struct. Mol. Biol. 13:1084‐1091.
   Zhou, Z. , Zhen, J. , Karpowich, N.K. , Goetz, R.M. , Law, C.J. , Reith, M.E. , and Wang, D.N. 2007. LeuT‐desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317:1390‐1393.
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