Metal‐Chelate Affinity Chromatography

Kevin J. Petty1

1 Merck & Company, West Point, Pennsylvania
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.10
DOI:  10.1002/0471142301.ns0510s05
Online Posting Date:  May, 2001
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Abstract

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxy terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique is know as metal‐chelate affinity chromatography and can be performed using either native or denatured protein. This unit presents protocols for expression of histidine‐tail fusion proteins and their purification in either native or denatured form (along with procedures for renaturation by either dialysis or solid‐phase renaturation). Also provided are procedures for analysis of the purified produce and regeneration of the NTA resin.

     
 
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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
  • M9ZB 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
  • MCAC‐0, MCAC‐20, MCAC‐40, MCAC‐60, MCAC‐80, MCAC‐100, MCAC‐200, and MCAC‐1000 buffers (see recipe)
  • 150× 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 (see CPMB UNIT and appendix 1A in this manual) 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)

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

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

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 see protocol 2Alternate Protocols 1 or protocol 32)
  • 2× SDS sample buffer ( appendix 2A)
  • MCAC‐0 buffer (see recipe)
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (see CPMB UNIT and appendix 1A in this manual), and cleavage of proteins with factor Xa or thrombin (Wingfield et al., )

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 ( appendix 2A)
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Figures

Videos

Literature Cited

Literature Cited
   Andersson, L. and Porath, J. 1986. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 154:250‐254.
   Botting, C.H. and Randall, R.E. 1995. Reporter enzyme–nitrilotriacetic acid–nickel conjugates: Reagents for detecting histidine‐tagged proteins. Biotechniques 19:362‐363.
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  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.
   Hochuli, E., Dobeli, H., and Schacher, A. 1987. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues J. Chromatogr. 411:177‐184.
  Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. and Stüber, D. 1988. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent Bio/Technology 6:1321‐1325.
   Hoffmann, A. and Roeder, R. 1991. Purification of His‐tagged proteins in nondenaturing conditions suggests a convenient method for protein interaction studies. Nucl. Acids Res. 19:6337‐6338.
   Hutchens, T.W. and Yip, T.‐T. 1990a. Protein interactions with immobilized transition metal ions: Quantitative evaluations of variations in affinity and binding capacity. Anal. Biochem. 191:160‐168.
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   Janknecht, R. and Nordheim, A. 1992. Affinity purification of histidine‐tagged proteins transiently produced in HeLa cells. Gene. 121:321‐324.
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   Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation Nature 258:598‐599.
   Struhl, K. 1987. Subcloning of DNA fragments. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.16.1‐3.16.11. John Wiley & Sons, New York.
   Stüber, D., Matile, H., and Garotta, G. 1990. System for high‐level production in Escherichia coli and rapid purification of recombinant proteins: Application to epitope mapping, preparation of antibodies, and structure‐function analysis. Immunol. Methods 4:121‐152.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 polymerase to direct expression of cloned genes. Methods Enzymol. 185:60‐89.
   Taussig, R., Quarmby, L., and Gilman, A. 1993. Regulation of purified type I and type II adenylylcyclases by G protein β‐γ subunits. J. Biol. Chem. 268:9‐12.
   Wingfield, P.T., Palmer, I., and Liang, S.‐M. 1995. Folding and purification of insoluble (inclusion‐body) proteins from Escherichia coli. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds.) pp. 6.5.1‐6.5.27. John Wiley & Sons, New York.
   Yip, T.‐T., Nakagawa, Y. and Porath, J. 1989. Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high‐performance immobilized metal ion affinity chromatography. Anal. Biochem. 183:159‐171.
Key Reference
   Hochuli, 1990. See above.
  Describes basic principles of MCAC with detailed protocols.
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