Arrestin Expression in E. coli and Purification

Sergey A. Vishnivetskiy1, Xuanzhi Zhan1, Qiuyan Chen1, Tina M. Iverson1, Vsevolod V. Gurevich1

1 Department of Pharmacology, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.11
DOI:  10.1002/0471141755.ph0211s67
Online Posting Date:  December, 2014
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Abstract

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag‐free wild‐type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin‐Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q‐Sepharose or SP‐Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological levels. © 2014 by John Wiley & Sons, Inc.

Keywords: arrestin; recombinant; expression; purification; chromatography

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Large‐Scale Expression and Purification of Arrestins
  • Alternate Protocol 1: Purification of Arrestin‐3 and Truncated Form of Arrestin‐1‐(1‐378)
  • Support Protocol 1: Small‐Scale Test Expression of Wild‐Type and Mutant Arrestins in E. coli
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Large‐Scale Expression and Purification of Arrestins

  Materials
  • LB broth (Sigma): prepare according to the manufacturer's instructions
  • 100 mg/ml ampicillin (Amp) stock solution in distilled water: store at 4oC
  • Cells transformed with arrestin expression constructs, glycerol stock (see Gurevich et al., ; Gurevich and Benovic, ): available from V.V. Gurevich upon request ( )
  • 100 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG): prepare in distilled water; store up to 2 years at −80°C
  • Lysis buffer (see recipe)
  • Lysozyme from chicken egg white (Sigma)
  • 1.0 M magnesium chloride solution (Sigma)
  • 15,000 U deoxyribonuclease I from bovine pancreas (DNase I; Sigma)/ml lysis buffer (see recipe)
  • 20,000 U deoxyribonuclease II from porcine spleen (DNase II; Sigma)/ml lysis buffer (see recipe)
  • 100 mg ribonuclease A from bovine pancreas (RNase A; Sigma)/ml lysis buffer (see recipe)
  • Ammonium sulfate
  • Multiple column buffer (MCB; see recipe)
  • Heparin‐Sepharose 6 Fast Flow (GE Healthcare)
  • 100 mM, 150 mM, 200 mM, 2 M, and 3 M NaCl in MCB (see recipe)
  • 100% (v/v) methanol
  • 2× SDS sample buffer (Sigma)
  • 10% (w/v) SDS‐PAGE gel
  • Q‐Sepharose Fast Flow (GE Healthcare)
  • SP‐Sepharose Fast Flow (GE Healthcare)
  • F431 rabbit polyclonal antibody (see Song et al., ): available from V.V. Gurevich upon request ( )
  • 500‐ml flasks
  • 2‐liter flasks
  • 30°C incubator with shaking platform
  • RC‐5B Plus centrifuge with SLA 3000 rotor (Sorvall)
  • 500‐ml centrifuge bottles
  • Sonicator (Fisher Dimembrator model 500, or equivalent)
  • TL‐100 tabletop ultracentrifuge with TLA 120.1 rotor (Beckman)
  • 1.5‐ml microcentrifuge tubes
  • 1‐liter beaker
  • Orbital shaker
  • 0.8‐μm syringe filter, sterile (Corning)
  • 1.5 × 20–cm Econo‐Column chromatography columns (Bio‐Rad, cat. no. 737‐1522)
  • GradiFrac chromatography system (Pharmacia Biotech)
  • Amicon filter unit with YM30 filters (Millipore)
  • Additional reagents and equipment for carrying out SDS‐PAGE electrophoresis ( appendix 3B; Gallagher and Sasse, ), western blotting (Gallagher et al., 2011; also see Gurevich et al., ; Gurevich and Benovic, ), and protein quantification ( appendix 3A; Olson and Markwell, )

Alternate Protocol 1: Purification of Arrestin‐3 and Truncated Form of Arrestin‐1‐(1‐378)

  Additional Materials
  • Heparin‐Sepharose arrestin fractions (from Basic Protocol, step 34)
  • 5.0 M NaCl solution (Sigma)

Support Protocol 1: Small‐Scale Test Expression of Wild‐Type and Mutant Arrestins in E. coli

  Additional Materials
  • 15‐ml conical, plastic tubes, sterile
  • 500‐μl centrifuge tube (Beckman)
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Figures

Videos

Literature Cited

Literature Cited
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