Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris

Shweta Singh1, Minghao Zhang1, Nicolas Bertheleme1, Philip G. Strange2, Bernadette Byrne1

1 Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom, 2 School of Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.4
DOI:  10.1002/0471140864.ps2904s67
Online Posting Date:  February, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The isolation of membrane proteins with the aim of producing highly pure, homogeneous, stable, and functional material remains challenging, and it is often necessary to develop protein‐specific purification protocols by trial and error. One key tool that is required in the development of a suitable protocol is a functional assay. This unit describes a range of different protocols for isolation of the human adenosine A2a receptor (A2aR). These protocols show the importance of developing a robust method for comparing the quality of protein obtained by a combination of biophysical analyses including SDS‐PAGE, analytical size‐exclusion chromatography, and functional analysis. One of the keys to isolating and maintaining a functional receptor, found not only in the optimal protocol described here but in other published examples, is that there should be no more than two chromatographic steps.

Keywords: G protein−coupled receptor; adenosine A2aR; immobilized metal affinity chromatography; size‐exclusion chromatography; ion exchange; antibody affinity chromatography; SDS‐PAGE; radioligand binding analysis; aggregation

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Adenosine A2aR by Antibody Affinity Chromatography Followed by TEV Cleavage and Reverse IMAC
  • Alternate Protocol 1: Immobilized Metal Affinity Chromatography
  • Alternate Protocol 2: Ion‐Exchange Chromatography
  • Basic Protocol 2: SDS‐PAGE and Immunoblotting Analysis
  • Basic Protocol 3: Analytical Size‐Exclusion Chromatography
  • Basic Protocol 4: Analysis of Receptor Sensitivity to Degradation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Purification of Adenosine A2aR by Antibody Affinity Chromatography Followed by TEV Cleavage and Reverse IMAC

  Materials
  • 15 to 20 mg solubilized membrane protein containing ∼2 mg receptor (unit 29.3)
  • M2 equilibration buffer (see recipe)
  • M2 affinity resin (Sigma‐Aldrich)
  • M2 affinity buffer (see recipe)
  • FLAG peptide (Sigma‐Aldrich)
  • 6His‐TEV protease (see recipe)
  • Dialysis buffer (see recipe)
  • IMAC buffer (see recipe)
  • Imidazole (Sigma‐Aldrich)
  • Drip column: 25‐ml Poly‐Prep/Glass Econo‐column (Bio‐Rad)
  • 50‐ml Falcon tube
  • Rolling shaker
  • Dialysis tubing, 12‐ to 14‐kDa molecular weight cutoff (MWCO; Spectrumlabs)
  • 5‐ml His‐trap column (GE Lifesciences)
  • Peristaltic pump P‐1 (GE Lifesciences)
  • 100‐kDa MWCO concentrator (Amicon; optional)
  • Additional reagents and equipment for preparation of solubilized receptor (unit 29.3)
NOTE: All steps are carried out at 4°C.

Alternate Protocol 1: Immobilized Metal Affinity Chromatography

  Materials
  • 15 to 20 mg solubilized membrane protein containing ∼2 mg receptor (unit 29.3)
  • IMAC resin: Co2+‐TALON resin (Clontech) or Ni2+‐NTA superflow resin (Qiagen)
  • IMAC buffer (see recipe)
  • IMAC wash buffer (see recipe)
  • IMAC elution buffer (see recipe)
  • 50‐ml Falcon tube
  • Rolling shaker
  • Drip column: 25‐ml Poly‐Prep/Glass Econo‐column (Bio‐Rad)
NOTE: All steps are carried out at 4°C.

Alternate Protocol 2: Ion‐Exchange Chromatography

  Materials
  • Partially purified protein (see protocol 1 or protocol 2)
  • MonoQ buffer (see recipe)
  • MonoQ elution buffer (see recipe)
  • 100‐kDa MWCO concentrators (Amicon)
  • 1‐ml MonoQ 5/5 gl columns (GE Lifesciences)
  • Akta FPLC system (GE Lifesciences)

Basic Protocol 2: SDS‐PAGE and Immunoblotting Analysis

  Materials
  • Purified protein (see protocol 1 or protocol 2 or protocol 3)
  • SDS‐loading dye (Invitrogen)
  • 4‐12% Bis‐Tris gels (Invitrogen)
  • MES gel running buffer (Invitrogen)
  • Imperial stain (Thermo Fisher; optional)
  • Methanol HiPerSolv (BDH)
  • Protein transfer buffer (Invitrogen)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Skimmed milk powder (Marvel)
  • Primary antibody: mouse M2 anti‐FLAG antibody (Sigma‐Aldrich) or anti‐His antibody (Dianova)
  • Tween‐20 (Promega)
  • Secondary antibody: horseradish peroxidase (HRP)− or alkaline phosphatase (AP)−conjugated anti−mouse IgG (Sigma‐Aldrich)
  • ECL or ECF substrate (GE Lifesciences)
  • PVDF membrane (Millipore)
  • LAS‐1000‐3000 charged‐coupled device (CCD) imaging system (Fujifilm)
  • Additional reagents and equipment for immunoblotting (unit 10.7) and detection (unit 10.10)
NOTE: All steps are carried out at room temperature unless otherwise indicated.

Basic Protocol 3: Analytical Size‐Exclusion Chromatography

  Materials
  • Purified protein (see protocol 1 or protocol 2 or protocol 3)
  • SEC buffer (see recipe)
  • Superose 6 10/300 GL column (GE Lifesciences)
  • Akta FPLC system (GE Lifesciences)

Basic Protocol 4: Analysis of Receptor Sensitivity to Degradation

  Materials
  • Purified protein (see protocol 1 or protocol 2 or protocol 3)
  • SDS‐loading dye (Invitrogen)
  • Liquid nitrogen
  • Additional reagents and equipment for SDS‐PAGE or immunoblotting (see protocol 4)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
   Alguel, Y., Leung, J., Singh, S., Rana, R., Civiero, L., Alves, C., and Byrne, B. 2010. New tools for membrane protein research. Curr. Protein Pept. Sci. 11:156‐165.
   Chae, P.S., Rasmussen, S.G., Rana, R.R., Gotfryd, K., Chandra, R., Goren, M.A., Kruse, A.C., Nurva, S., Loland, C.J., Pierre, Y., Drew, D., Popot, J.L., Picot, D., Fox, B.G., Guan, L., Gether, U., Byrne, B., Kobilka, B., and Gellman, S.H. 2010. Maltose‐neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7:1003‐1008.
   Chen, C.C. and Wilson, T.H. 1984. The phospholipid requirement for activity of the lactose carrier of Escherichia coli. J. Biol. Chem. 259:10150‐10158.
   Cherezov, V., Rosenbaum, D.M., Hanson, M.A., Rasmussen, S.G., Thian, F.S., Kobilka, T.S., Choi, H.J., Kuhn, P., Weis, W.I., Kobilka, B.K., and Stevens, R.C. 2007. High‐resolution crystal structure of an engineered human beta2‐adrenergic G protein‐coupled receptor. Science 318:1258‐1265.
   Chien, E.Y., Liu, W., Zhao, Q., Katritch, V., Han, G.W., Hanson, M.A., Shi, L., Newman, A.H., Javitch, J.A., Cherezov, V., and Stevens, R.C. 2010. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330:1091‐1095.
   Chiu, M.L., Tsang, C., Grihalde, N., and MacWilliams, M.P. 2008. Over‐expression, solubilization, and purification of G protein‐coupled receptors for structural biology. Comb. Chem. High Throughput Screen. 11:439‐462.
   Fraser, N.J. 2006. Expression and functional purification of a glycosylation deficient version of the human adenosine 2a receptor for structural studies. Protein Expr. Purif. 49:129‐137.
   Grisshammer, R. and Tate, C.G. 1995. Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28:315‐422.
   Gutmann, D.A., Mizohata, E., Newstead, S., Ferrandon, S., Postis, V., Xia, X., Henderson, P.J., van Veen, H.W., and Byrne, B. 2007. A high‐throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay. Protein Sci. 16:1422‐1428.
   Hanson, M.A., Cherezov, V., Griffith, M.T., Roth, C.B., Jaakola, V.P., Chien, E.Y., Velasquez, J., Kuhn, P., and Stevens, R.C. 2008. A specific cholesterol binding site is established by the 2.8 A structure of the human beta2‐adrenergic receptor. Structure 16:897‐905.
   Hein, L. and Kobilka, B.K. 1995. Adrenergic receptor signal transduction and regulation. Neuropharmacology 34:357‐366.
   Jaakola, V.P., Griffith, M.T., Hanson, M.A., Cherezov, V., Chien, E.Y., Lane, J.R., Ijzerman, A.P., and Stevens, R.C. 2008. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322:1211‐1217.
   Lucast, L.J., Batey, R.T., and Doudna, J.A. 2001. Large‐scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544‐546, 548, 550 passim.
   McCusker, E.C., Bane, S.E., O'Malley, M.A., and Robinson, A.S. 2007. Heterologous GPCR expression: A bottleneck to obtaining crystal structures. Biotechnol. Prog. 23:540‐547.
   Rasmussen, S.G., Choi, H.J., Rosenbaum, D.M., Kobilka, T.S., Thian, F.S., Edwards, P.C., Burghammer, M., Ratnala, V.R., Sanishvili, R., Fischetti, R.F., Schertler, G.F., Weis, W.I., and Kobilka, B.K. 2007. Crystal structure of the human beta2 adrenergic G‐protein‐coupled receptor. Nature 450:383‐387.
   Rath, A., Glibowicka, M., Nadeau, V.G., Chen, G., and Deber, C.M. 2009. Detergent binding explains anomalous SDS‐PAGE migration of membrane proteins. Proc. Natl. Acad. Sci. U.S.A. 106:1760‐1765.
   Rosenbaum, D.M., Cherezov, V., Hanson, M.A., Rasmussen, S.G., Thian, F.S., Kobilka, T.S., Choi, H.J., Yao, X.J., Weis, W.I., Stevens, R.C., and Kobilka, B.K. 2007. GPCR engineering yields high‐resolution structural insights into beta2‐adrenergic receptor function. Science 318:1266‐1273.
   Sarramegna, V., Talmont, F., Demange, P., and Milon, A. 2003. Heterologous expression of G‐protein‐coupled receptors: Comparison of expression systems fron the standpoint of large‐scale production and purification. Cell. Mol. Life Sci. 60:1529‐1546.
   Sharma, S.K., Evans, D.B., Vosters, A.F., Chattopadhyay, D., Hoogerhelde, J.G., and Campbell, C.M. 1992. Immobilized metal affinity chromatography of bacterially expressed protein engineered to contain an alternating‐histidine domain. Methods 4:57‐67.
   Singh, S., Gras, A., Fiez‐Vandal, C., Ruprecht, J., Rana, R., Martinez, M., Strange, P.G., Wagner, R., and Byrne, B. 2008. Large‐scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures. Microb. Cell. Fact. 7:28.
   Singh, S., Hedley, D., Kara, E., Gras, A., Iwata, S., Ruprecht, J., Strange, P.G., and Byrne, B. 2010. A purified C‐terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant. Protein Expr. Purif. 74:80‐87.
   Singh, S., Gras, A., Fiez‐Vandal, C., Martinez, M., Wagner, R., and Byrne, B. 2012. Large scale production of membrane proteins in Pichia pastoris: The production of G protein‐coupled receptors as a case study. Meth. Mol. Biol., in press.
   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.
   Strop, P. and Brunger, A.T. 2005. Refractive index‐based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14:2207‐2211.
   Warne, T., Chirnside, J., and Schertler, G.F. 2003. Expression and purification of truncated, non‐glycosylated turkey beta‐adrenergic receptors for crystallization. Biochim. Biophys. Acta 1610:133‐140.
   Warne, T., Serrano‐Vega, M.J., Baker, J.G., Moukhametzianov, R., Edwards, P.C., Henderson, R., Leslie, A.G., Tate, C.G., and Schertler, G.F. 2008. Structure of a beta1‐adrenergic G‐protein‐coupled receptor. Nature 454:486‐491.
   Wu, B., Chien, E.Y., Mol, C.D., Fenalti, G., Liu, W., Katritch, V., Abagyan, R., Brooun, A., Wells, P., Bi, F.C., Hamel, D.J., Kuhn, P., Handel, T.M., Cherezov, V., and Stevens, R.C. 2010. Structures of the CXCR4 chemokine GPCR with small‐molecule and cyclic peptide antagonists. Science 330:1066‐1071.
Key References
   The following manuals provide a good starting point for protocol development and troubleshooting for IMAC and FLAG tag purification, respectively.
  http://www.clontech.com/images/pt/PT1320‐1.pdf
   The following handbook provides a general overview on Purification of Challenging Proteins.
  http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/a2220bul.Par.0001.File.tmp/a2220bul.pdf
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library