Protein Purification Using PDZ Affinity Chromatography

Ward G. Walkup1, Mary B. Kennedy1

1 Department of Biology and Biological Engineering, California Institute of Technology, Pasadena, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.10
DOI:  10.1002/0471140864.ps0910s80
Online Posting Date:  April, 2015
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PDZ domains function in nature as protein‐binding domains within scaffold and membrane‐associated proteins. They comprise approximately 90 residues and undergo specific, high‐affinity interactions with complementary C‐terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain‐binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. © 2015 by John Wiley & Sons, Inc.

Keywords: affinity chromatography; protein purification; affinity tag; peptide; PDZ domain; matrices; ligand

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of PDZ Ligand Affinity Resin
  • Basic Protocol 2: Preparation of PDZ Domain Affinity Resin
  • Alternate Protocol 1: Preparation of PDZ Domain Affinity Resin Using HaloTag Technology
  • Basic Protocol 3: Purification of POIs Using PDZ Affinity Chromatography
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Preparation of PDZ Ligand Affinity Resin

  • NHS‐Activated Agarose (Life Technologies, cat. no. 26200)
  • Ultrapure H 2O
  • Coupling/wash buffer I (see recipe)
  • 300 mg PDZ ligand peptide (75% purity or above; e.g., GAGSSIESDV)
  • Quenching buffer (see recipe)
  • Storage buffer I (see recipe)
  • 50 ml screw‐cap polypropylene tubes (e.g., BD Falcon), sterile
  • Centrifuge
  • End‐over‐end or rotating wheel mixer
  • Additional reagents and equipment for determining peptide concentration (unit 3.4; Olson and Markwell, )

Basic Protocol 2: Preparation of PDZ Domain Affinity Resin

  • BL21(DE3) E. coli cells (e.g., Life Technologies)
  • Expression plasmid containing PDZ domain cDNA (Walkup and Kennedy, )
  • LB Miller Agar (Teknova)
  • Carbenicillin or other appropriate selection antibiotic
  • LB Miller Broth (Teknova)
  • 1 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • BugBuster lysis buffer (see recipe)
  • Purification buffer (see recipe)
  • Benzonase (e.g., Sigma‐Aldrich; optional)
  • ReadyLyse (; optional)
  • Prepared PDZ ligand affinity resin (see protocol 1)
  • Peptide elution buffer (see recipe)
  • Denaturing buffer (see recipe)
  • Storage buffer II (see recipe)
  • Coupling/wash buffer II (see recipe)
  • NHS‐Activated Agarose (Life Technologies, cat. no. 26200)
  • Ultrapure H 2O
  • Quenching buffer (see recipe)
  • 14‐ml conical tubes (e.g., BD Falcon)
  • Orbital shaker
  • 2‐liter plastic baffled Erlenmeyer flasks
  • Spectrophotometer
  • Refrigerated centrifuge
  • Teflon‐glass or Dounce homogenizer
  • End‐over‐end or rotating wheel mixer
  • Microfluidizer (e.g.,; optional)
  • 50‐ml conical tubes (e.g., BD Falcon)
  • Plastic or glass chromatography columns
  • Protein concentrators, 9 kDa molecular weight cutoff
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, )

Alternate Protocol 1: Preparation of PDZ Domain Affinity Resin Using HaloTag Technology

  Additional Materials (also see protocol 2)
  • pFN18A plasmid (Promega, cat. no. G2751) containing HaloTag‐PDZ domain fusion protein (Walkup and Kennedy, )
  • Overnight Express Instant Terrific Broth (EMD Millipore)
  • HaloTag lysis buffer (see recipe)
  • HaloLink resin (Promega)
  • HaloTag purification buffer (see recipe)
  • HaloTag storage buffer (see recipe)
  • HaloTag cleavage buffer (see recipe) with and without TEV protease (Sigma or Promega)
  • 0.6‐ml microcentrifuge tubes
  • Additional reagents and equipment for Coomassie blue staining of gels (Echan and Speicher, )

Basic Protocol 3: Purification of POIs Using PDZ Affinity Chromatography

  • PDZ affinity resin (see Basic Protocol protocol 11 or protocol 22 or protocol 3Alternate Protocol)
  • Storage buffer I (optional; see recipe) and storage buffer II (see recipe)
  • Purification buffer (see recipe), or other column buffer
  • Partially purified POI or clarified lysate containing POI in purification buffer (see recipe) or other column buffer
  • Peptide elution buffer (see recipe)
  • Denaturing buffer (see recipe)
  • 50‐ml conical centrifuge tubes
  • End‐over‐end mixer
  • Protein concentrators
  • Refrigerated centrifuge
  • Disposable chromatography column
  • Liquid nitrogen
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Literature Cited

Literature Cited
  Adams, M.E., Butler, M.H., Dwyer, T.M., Peters, M.F., Murnane, A.A., and Froehner, S.C. 1993. Two forms of mouse syntrophin, a 58 kd dystrophin‐associated protein, differ in primary structure and tissue distribution. Neuron 11:531‐540.
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds). 2015. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
  Baneyx, F. and Mujacic, M. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nat. Biotechnol. 22:1399‐1408.
  Bolanos‐Garcia, V.M. and Davies, O.R. 2006. Structural analysis and classification of native proteins from E. coli commonly co‐purified by immobilised metal affinity chromatography. Biochim. Biophys. Acta 1760:1304‐1313.
  Brenman, J.E., Chao, D.S., Gee, S.H., McGee, A.W., Craven, S.E., Santillano, D.R., Wu, Z., Huang, F., Xia, H., Peters, M.F., Froehner, S.C., and Bredt, D.S. 1996. Interaction of nitric oxide synthase with the postsynaptic density protein PSD‐95 and alpha1‐syntrophin mediated by PDZ domains. Cell 84:757‐767.
  Cho, K.O., Hunt, C.A., and Kennedy, M.B. 1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs‐large tumor suppressor protein. Neuron 9:929‐942.
  di Guan, C., Li, P., Riggs, P.D., and Inouye, H. 1988. Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose‐binding protein. Gene 67:21‐30.
  Dorn, I.T., Neumaier, K.R., and Tampé, R. 1998. Molecular recognition of histidine‐tagged molecules by metal‐chelating lipids monitored by fluorescence energy transfer and correlation spectroscopy§. J. Am. Chem. Soc. 120:2753‐2763.
  Doyle, D.A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. 1996. Crystal structures of a complexed and peptide‐free membrane protein‐binding domain: Molecular basis of peptide recognition by PDZ. Cell 85:1067‐1076.
  Echan, L.A. and Speicher, D. W. 2002. Protein detection in gels using fixation. Curr. Protoc. Protein Sci. 29:10.5:10.5.1‐10.5.18.
  Fabrini, R., De Luca, A., Stella, L., Mei, G., Orioni, B., Ciccone, S., Federici, G., Lo Bello, M., and Ricci, G. 2009. Monomer‐dimer equilibrium in glutathione transferases: A critical re‐examination. Biochemistry 48:10473‐10482.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44.
  Gee, S.H., Sekely, S.A., Lombardo, C., Kurakin, A., Froehner, S.C., and Kay, B.K. 1998. Cyclic peptides as non‐carboxyl‐terminal ligands of syntrophin PDZ domains. J. Biol. Chem. 273:21980‐21987.
  Gibson, D.G. 2011. Enzymatic assembly of overlapping DNA fragments. Meth. Enzymol. 498:349‐361.
  Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd, and Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6:343‐345.
  Grandy, D., Shan, J., Zhang, X., Rao, S., Akunuru, S., Li, H., Zhang, Y., Alpatov, I., Zhang, X.A., Lang, R.A., Shi, D.L., and Zheng, J.J. 2009. Discovery and characterization of a small molecule inhibitor of the PDZ domain of dishevelled. J. Biol. Chem. 284:16256‐16263.
  Guignet, E.G., Hovius, R., and Vogel, H. 2004. Reversible site‐selective labeling of membrane proteins in live cells. Nat. Biotechnol. 22:440‐444.
  Harris, B.Z., Hillier, B.J., and Lim, W.A. 2001. Energetic determinants of internal motif recognition by PDZ domains. Biochemistry 40:5921‐5930.
  Hermanson, G.T. 2013. Bioconjugate techniques, Third edition. Elsevier/AP, London.
  Hillier, B.J., Christopherson, K.S., Prehoda, K.E., Bredt, D.S., and Lim, W.A. 1999. Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS‐syntrophin complex. Science 284:812‐815.
  Hung, A.Y. and Sheng, M. 2002. PDZ domains: Structural modules for protein complex assembly. J. Biol. Chem. 277:5699‐5702.
  Ikura, K., Kokubu, T., Natsuka, S., Ichikawa, A., Adachi, M., Nishihara, K., Yanagi, H., and Utsumi, S. 2002. Co‐overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli. Prep. Biochem. Biotechnol. 32:189‐205.
  Ivanetich, K.M. and Goold, R.D. 1989. A rapid equilibrium random sequential bi‐bi mechanism for human placental glutathione S‐transferase. Biochim. Biophys. Acta 998:7‐13.
  Ivanetich, K.M., Thumser, A.E., and Harrison, G.G. 1988. Halothane: Inhibition and activation of rat hepatic glutathione S‐transferases. Biochem. Pharmacol. 37:1903‐1908.
  Ivanetich, K.M., Goold, R.D., and Sikakana, C.N. 1990. Explanation of the non‐hyperbolic kinetics of the glutathione S‐transferases by the simplest steady‐state random sequential Bi Bi mechanism. Biochem. Pharmacol. 39:1999‐2004.
  Ivarsson, Y., Wawrzyniak, A.M., Wuytens, G., Kosloff, M., Vermeiren, E., Raport, M., and Zimmermann, P. 2011. Cooperative phosphoinositide and peptide binding by PSD‐95/discs large/ZO‐1 (PDZ) domain of polychaetoid, Drosophila zonulin. J. Biol. Chem. 286:44669‐44678.
  Jana, S. and Deb, J.K. 2005. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67:289‐298.
  Khan, F., He, M., and Taussig, M.J. 2006. Double‐hexahistidine tag with high‐affinity binding for protein immobilization, purification, and detection on Ni‐nitrilotriacetic acid surfaces. Anal. Chem. 78:3072‐3079.
  Kimple, M.E. and Sondek, J. 2002. Affinity tag for protein purification and detection based on the disulfide‐linked complex of InaD and NorpA. BioTechniques 33:578, 580, 584‐578 passim.
  Kimple, M.E., Brill, A.L., and Pasker, R.L. 2013. Overview of affinity tags for protein purification. Curr. Protoc. Protein. Sci. 73:9.9.1‐9.9.23.
  Klock, H.E. and Lesley, S.A. 2009. The Polymerase Incomplete Primer Extension (PIPE) method applied to high‐throughput cloning and site‐directed mutagenesis. Methods Mol. Biol. 498:91‐103.
  Klock, H.E., Koesema, E.J., Knuth, M.W., and Lesley, S.A. 2008. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982‐994.
  Kornau, H.C., Seeburg, P.H., and Kennedy, M.B. 1997. Interaction of ion channels and receptors with PDZ domain proteins. Curr. Opin. Neurobiol. 7:368‐373.
  Kornau, H.C., Schenker, L.T., Kennedy, M.B., and Seeburg, P.H. 1995. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD‐95. Science 269:1737‐1740.
  Ladunga, I. 2009. Finding homologs in amino acid sequences using network BLAST searches. Curr. Protoc. Bioinform. 25:3.4.1‐3.4.34.
  Lata, S., Reichel, A., Brock, R., Tampe, R., and Piehler, J. 2005. High‐affinity adaptors for switchable recognition of histidine‐tagged proteins. J. Am. Chem. Soc. 127:10205‐10215.
  Lim, I.A., Hall, D.D., and Hell, J.W. 2002. Selectivity and promiscuity of the first and second PDZ domains of PSD‐95 and synapse‐associated protein 102. J. Biol. Chem. 277:21697‐21711.
  Lim, I.A., Merrill, M.A., Chen, Y., and Hell, J.W. 2003. Disruption of the NMDA receptor‐PSD‐95 interaction in hippocampal neurons with no obvious physiological short‐term effect. Neuropharmacology 45:738‐754.
  Los, G.V. and Wood, K. 2007. The HaloTag: A novel technology for cell imaging and protein analysis. Methods Mol. Biol. 356:195‐208.
  Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., Simpson, D., Mendez, J., Zimmerman, K., Otto, P., Vidugiris, G., Zhu, J., Darzins, A., Klaubert, D.H., Bulleit, R.F., and Wood, K.V. 2008. HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3:373‐382.
  Luong, J.H. and Scouten, W.H. 2008. Affinity purification of natural ligands. Curr. Protoc. Protein Sci. 52:9.3.1‐9.3.22.
  Maina, C.V., Riggs, P.D., Grandea, A.G., 3rd, Slatko, B.E., Moran, L.S., Tagliamonte, J.A., McReynolds, L.A., and Guan, C.D. 1988. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose‐binding protein. Gene 74:365‐373.
  Makrides, S.C. 1996. Strategies for achieving high‐level expression of genes in Escherichia coli. Microbiol. Rev. 60:512‐538.
  Malhotra, A. 2009. Tagging for protein expression. Meth. Enzymol. 463:239‐258.
  Marfatia, S.M., Morais Cabral, J.H., Lin, L., Hough, C., Bryant, P.J., Stolz, L., and Chishti, A.H. 1996. Modular organization of the PDZ domains in the human discs‐large protein suggests a mechanism for coupling PDZ domain‐binding proteins to ATP and the membrane cytoskeleton. J. Cell Biol. 135:753‐766.
  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.
  Muller, B.M., Kistner, U., Kindler, S., Chung, W.J., Kuhlendahl, S., Fenster, S.D., Lau, L.F., Veh, R.W., Huganir, R.L., Gundelfinger, E.D., and Garner, C.C. 1996. SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo. Neuron 17:255‐265.
  Nieba, L., Nieba‐Axmann, S.E., Persson, A., Hamalainen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F., and Pluckthun, A. 1997. BIACORE analysis of histidine‐tagged proteins using a chelating NTA sensor chip. Anal. Biochem. 252:217‐228.
  Niethammer, M., Valtschanoff, J.G., Kapoor, T.M., Allison, D.W., Weinberg, R.J., Craig, A.M., and Sheng, M. 1998. CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD‐95/SAP90. Neuron 20:693‐707.
  Nishihara, K., Kanemori, M., Yanagi, H., and Yura, T. 2000. Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Appl. Environ. Microbiol. 66:884‐889.
  Notredame, C. 2010. Computing multiple sequence/structure alignments with the T‐coffee package. Curr. Protoc. Bioinform. 29:3.8:3.8.1‐3.8.25.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29.
  Qing, G., Ma, L.C., Khorchid, A., Swapna, G.V., Mal, T.K., Takayama, M.M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M., and Inouye, M. 2004. Cold‐shock induced high‐yield protein production in Escherichia coli. Nat. Biotechnol. 22:877‐882.
  Robichon, C., Luo, J., Causey, T.B., Benner, J.S., and Samuelson, J.C. 2011. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl. Environ. Microbiol. 77:4634‐4646.
  Sambrook, J., Maniatis, T., and Fritsch, E.F. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  Saro, D., Li, T., Rupasinghe, C., Paredes, A., Caspers, N., and Spaller, M.R. 2007. A thermodynamic ligand binding study of the third PDZ domain (PDZ3) from the mammalian neuronal protein PSD‐95. Biochemistry 46:6340‐6352.
  Schein, C.H. 1989. Production of soluble recombinant proteins in bacteria. Biotechnology 7:1141‐1147.
  Seedorff, S., Appelt, C., Beyermann, M., and Schmieder, P. 2010. Design, synthesis, structure and binding properties of PDZ binding, cyclic beta‐finger peptides. Biochem. Biophys. Res. Commun. 395:535‐539.
  Simossis, V., Kleinjung, J., and Heringa, J. 2003. An overview of multiple sequence alignment. Curr. Protoc. Bioinforms. 3:3.7.1‐3.7.26.
  Smith, D.B. 2000. Generating fusions to glutathione S‐transferase for protein studies. Meth. Enzymol. 326:254‐270.
  Smith, D.B. and Johnson, K.S. 1988. Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S‐transferase. Gene 67:31‐40.
  Songyang, Z., Fanning, A.S., Fu, C., Xu, J., Marfatia, S.M., Chishti, A.H., Crompton, A., Chan, A.C., Anderson, J.M., and Cantley, L.C. 1997. Recognition of unique carboxyl‐terminal motifs by distinct PDZ domains. Science 275:73‐77.
  Sorensen, H.P. and Mortensen, K.K. 2005. Advanced genetic strategies for recombinant protein expression in Escherichia coli. J. Biotechnol. 115:113‐128.
  Tochio, H., Zhang, Q., Mandal, P., Li, M., and Zhang, M. 1999. Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide. Nat. Struct. Biol. 6:417‐421.
  Vera, A., Gonzalez‐Montalban, N., Aris, A., and Villaverde, A. 2007. The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol. Bioeng. 96:1101‐1106.
  Walkup IV, W.G. and Kennedy, M.B. 2014. PDZ affinity chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands. Protein Expr. Purif. 98:46‐62.
  Wang, L., Piserchio, A., and Mierke, D.F. 2005. Structural characterization of the intermolecular interactions of synapse‐associated protein‐97 with the NR2B subunit of N‐methyl‐d‐aspartate receptors. J. Biol. Chem. 280:26992‐26996.
  Webb, B. and Sali, A. 2014. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 47:5.6.1‐5.6.32.
  Welch, M., Villalobos, A., Gustafsson, C., and Minshull, J. 2009b. You're one in a googol: Optimizing genes for protein expression. J.R. Soc. Interface 6 Suppl 4:S467‐476.
  Welch, M., Villalobos, A., Gustafsson, C., and Minshull, J. 2011. Designing genes for successful protein expression. Meth. Enzymol. 498:43‐66.
  Welch, M., Govindarajan, S., Ness, J.E., Villalobos, A., Gurney, A., Minshull, J., and Gustafsson, C. 2009a. Design parameters to control synthetic gene expression in Escherichia coli. PloS one 4:e7002.
  Young, C.L., Britton, Z.T., and Robinson, A.S. 2012. Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Plant. Biotechnol. J. 7:620‐634.
Key Reference
  Walkup and Kennedy, 2014. See above.
  Development of PDZ affinity chromatography methodology, including associated affinity tags and resin and purification of heterologously expressed neuronal proteins containing endogenous PDZ domains or ligands.
  Kimple and Sondek, 2002. See above.
  First paper to describe affinity chromatography using a PDZ domain and a peptide ligand. Unlike the PDZ domains and ligands utilized in Walkup and Kennedy (), here, the PDZ domain (InaD) and peptide ligand (NorpA) form a covalent complex that is sensitive to reducing agents such as DTT or TCEP.
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