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Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography

  • Naveen Kumar Singh
  • Roy N. DSouza
  • Noor Shad Bibi
  • Marcelo Fernández-LahoreEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1286)

Abstract

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Key words

Protein purification Graft-copolymerization Megaporous cryogels IMAC Affinity chromatography 

Notes

Acknowledgements

The authors thank Prof. Matthias S. Ullrich for providing cells carrying the Ncr gene for the production of the His6-tagged protein. M.F.L. is a member of the National Council for Science and Technology, Buenos Aires, Argentina. This work was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 312004.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Naveen Kumar Singh
    • 1
  • Roy N. DSouza
    • 1
  • Noor Shad Bibi
    • 1
  • Marcelo Fernández-Lahore
    • 1
    Email author
  1. 1.Downstream Bioprocessing Laboratory, School of Engineering and ScienceJacobs UniversityBremenGermany

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