Preparation of HCV NS3 and NS5B Proteins to Support Small‐Molecule Drug Discovery

Magdeleine Hung1, Ruth Wang1, Xiaohong Liu1

1 Gilead Sciences, Foster City, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13B.6
DOI:  10.1002/0471141755.ph13b06s54
Online Posting Date:  September, 2011
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Abstract

Production of high‐quality, well‐characterized recombinant proteins facilitates screening of compound libraries. The protocols detailed in this unit are used to purify three recombinant enzymes that are widely used in HCV research: the HCV NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B RNA‐dependent RNA polymerase. The active enzymes are purified to homogeneity by two‐column chromatography to support a screening program for HCV inhibitors. Curr. Protoc. Pharmacol. 54:13B.6.1‐13B.6.18. © 2011 by John Wiley & Sons, Inc.

Keywords: HCV; protease; helicase; polymerase; protein purification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of His‐Tagged HCV NS3(181) by HisTrap Affinity Chromatography
  • Basic Protocol 2: Purification of Full‐Length NS3+NS4A Complex Using SUMO Fusion
  • Basic Protocol 3: Purification of His‐Tagged HCV NS5BΔC21‐C6H by HisTrap Affinity Purification
  • Support Protocol 1: Cleaning HisTrap HP Columns
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of His‐Tagged HCV NS3(181) by HisTrap Affinity Chromatography

  Materials
  • Pellet of E. coli strain expressing a His 6‐tagged NS3 GT1b (181) construct (cloned in‐house; see Kim et al., )
  • NS3 lysis buffer (see recipe), 4°C
  • NS3 Ni column buffers A and B (see recipe), 4°C
  • NS3 SP buffers A and B (see recipe), 4°C
  • 1‐liter beaker
  • Homogenizer (e.g., Ultra‐Turrax T‐25 basic, Rose Scientific)
  • Cheese cloth
  • Microfluidizer (Microfluidics, Model M‐110L)
  • Refrigerated centrifuge and tubes (e.g., Avanti J‐26 XPI centrifuge [Beckman‐Coulter] with JA 25.5 rotor [cat. no. 363058] and 35‐ml tubes [cat. no. 361694])
  • 1‐liter bottle
  • ÄKTAexplorer 100 FPLC system (GE Healthcare)
  • 5‐ml HisTrap HP column (GE Healthcare, cat. no. 17‐5248‐01)
  • 5‐ml HiTrap SP‐HP column (GE Healthcare, cat. no. 17‐1151‐01)
  • 0.45‐µm filter (Corning)
  • Conductivity meter
  • NanoDrop 2000c spectrophotometer
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3B)
NOTE: The desired protein must be maintained cold throughout the isolation procedure. All reagents should be kept on ice and the FPLC should be maintained in a cold room (also see Critical Parameters and Troubleshooting).

Basic Protocol 2: Purification of Full‐Length NS3+NS4A Complex Using SUMO Fusion

  Materials
  • Pellet of E. coli strain expressing His 6‐SUMO‐FLNS3‐NS4A (con1b strain) construct (cloned in‐house; see Beran and Pyle, )
  • NS3‐4A lysis buffer (see recipe), 4°C
  • NS3‐4A Ni column buffers A and B (see recipe), 4°C
  • NS3‐4A dialysis buffer (see recipe), 4°C
  • NS3‐4A SUMO digestion buffer (see recipe), 4°C
  • SUMO protease (Invitrogen, cat. no. 12588‐018)
  • 5 M NaCl solution
  • NS3‐4A enzyme storage buffer (see recipe), 4°C
  • ÄKTAexplorer 100 FPLC system (GE Healthcare)
  • Three 5‐ml HisTrap HP columns (GE Healthcare, cat. no. 17‐5248‐01)
  • Conductivity meter
  • 10,000 MWCO dialysis cassettes (Thermo Scientific)
  • Additional reagents and equipment for preparation of bacterial lysate (see protocol 1), SDS‐PAGE ( appendix 3B), and determination of protein concentration ( appendix 3A)
NOTE: The desired protein must be maintained cold throughout the isolation procedure. All reagents should be kept on ice and the FPLC should be maintained in a cold room (also see Critical Parameters and Troubleshooting).

Basic Protocol 3: Purification of His‐Tagged HCV NS5BΔC21‐C6H by HisTrap Affinity Purification

  Materials
  • Pellet of E. coli strain expressing HCV NS5BΔC21‐C6H construct (cloned in‐house; see Ferrari et al., )
  • NS5B lysis buffer (see recipe), 4°C
  • NS5B Ni column buffers A and B (see recipe), 4°C
  • NS5B SP buffers A and B (see recipe), 4°C
  • Additional reagents and equipment described for purification of NS3 (see protocol 1)

Support Protocol 1: Cleaning HisTrap HP Columns

  Materials
  • 50 mM EDTA ( appendix 2A)
  • Milli‐Q‐purified water
  • 2 M NaCl
  • 1 M NaOH
  • 30% (v/v) isopropanol
  • 100 mM nickel chloride solution
  • 20% (v/v) ethanol
  • ÄKTAexplorer 100 FPLC system (GE Healthcare) with used HisTrap HP column
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Figures

Videos

Literature Cited

Literature Cited
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