Metal‐Chelate Affinity Chromatography

Kevin J. Petty1

1 University of Texas Southwestern Medical Center, Dallas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.4
DOI:  10.1002/0471140864.ps0904s04
Online Posting Date:  May, 2001
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Abstract

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal‐chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for xpression of histidine‐tail fusion proteins and their purification in native form by MCAC is described, and two alternate protocols describe purification of histidine‐tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid‐phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Native MCAC for Purification of Soluble Histidine‐Tail Fusion Proteins
  • Alternate Protocol 1: Denaturing MCAC for Purification of Insoluble Histidine‐Tail Fusion Proteins
  • Alternate Protocol 2: Solid‐Phase Renaturation of MCAC‐Purified Proteins
  • Support Protocol 1: Analysis and Processing of Purified Proteins
  • Support Protocol 2: NTA Resin Regeneration
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Native MCAC for Purification of Soluble Histidine‐Tail Fusion Proteins

  Materials
  • recipeM9ZB medium (see recipe) containing 50 µg/ml ampicillin and 25 µg/ml chloramphenicol
  • E. coli BL21(DE3)pLysS or other suitable strain (Novagen) containing a pET vector (Fig. ) expressing a histidine‐tail fusion protein
  • 0.1 M IPTG, filter sterilized
  • NTA resin slurry: 50% (w/v) suspension in 20% (v/v) ethanol (Qiagen)
  • 100 mM NiSO 4⋅6H 2O
  • recipeMCAC‐0, MCAC‐20, MCAC‐40, MCAC‐60, MCAC‐80, MCAC‐100, MCAC‐200, and MCAC‐1000 buffers (see recipe)
  • recipe150× protease inhibitor cocktail (see recipe)
  • 10% (v/v) Triton X‐100
  • 1 M MgCl 2
  • MCAC‐EDTA buffer (see recipe)
  • DNase I solution (see recipe)
  • Centrifuge with Beckman JA‐20 rotor or equivalent
  • 1 × 10–cm glass or polypropylene column
  • Additional reagents and equipment for growth of bacteria in liquid medium (unit 5.3) and analysis and processing of purified proteins by SDS‐PAGE (see protocol 4)

Alternate Protocol 1: Denaturing MCAC for Purification of Insoluble Histidine‐Tail Fusion Proteins

  • GuMCAC‐0, GuMCAC‐20, GuMCAC‐40, GuMCAC‐60, GuMCAC‐100, and GuMCAC‐500 buffers (see recipe)
  • GuMCAC‐EDTA buffer (see recipe)
  • Appropriate final buffer for protein (e.g., for proteolytic cleavage or long‐term storage)
  • Guanidine⋅HCl
  • Additional reagents and equipment for analysis and processing of purified proteins (see protocol 4) and dialysis ( appendix 3B & unit 4.4)

Alternate Protocol 2: Solid‐Phase Renaturation of MCAC‐Purified Proteins

  • 1:1 (v/v) MCAC‐20/GuMCAC‐20 buffer (see recipes)
  • 3:1 (v/v) MCAC‐20/GuMCAC‐20 buffer (see recipes)
  • 7:1 (v/v) MCAC‐20/GuMCAC‐20 buffer (see recipes)

Support Protocol 1: Analysis and Processing of Purified Proteins

  Materials
  • Fractions from MCAC column purification (crude extract, flowthroughs, and purified protein; see protocol 1 or protocol 2 or protocol 3)
  • 2× SDS sample buffer (unit 10.1)
  • MCAC‐0 buffer (see recipe)
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 10.1), cleavage of proteins with factor Xa or thrombin (unit 6.5), and dialysis (unit 4.4 & appendix 3B)

Support Protocol 2: NTA Resin Regeneration

  Materials
  • 2.5 ml used NTA resin (packed volume)
  • Stripping solution: 0.2 M acetic acid/6 M guanidine⋅HCl
  • 2% (w/v) SDS
  • 20%, 25%, 50%, 75%, and 100% (v/v) ethanol
  • 0.1 M EDTA, pH 8.0
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Figures

Videos

Literature Cited

Literature Cited
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Key Reference
   Hochuli, E. 1990. Purification of recombinant proteins with metal chelate adsorbent. In Genetic Engineering, Principles and Practice, Vol. 12 (J. Setlow, ed.) pp. 87‐98. Plenum, New York.
  Describes basic principles of MCAC with detailed protocols.
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