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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Abstract

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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Materials

Basic Protocol 1: Preparation of PDZ Ligand Affinity Resin

  Materials
  • 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

  Materials
  • 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 (http://www.epibio.com; 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., http://www.microfluidicscorp.com; 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

  Materials
  • 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

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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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