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

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Affinity Chromatography

Part of the book series: Methods in Molecular Biology ((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.

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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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Authors and Affiliations

  1. Downstream Bioprocessing Laboratory, School of Engineering and Science, Jacobs University, Campus Ring 1, Bremen, 28759, Germany

    Naveen Kumar Singh, Roy N. DSouza, Noor Shad Bibi & Marcelo Fernández-Lahore

Authors
  1. Naveen Kumar Singh
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  2. Roy N. DSouza
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  3. Noor Shad Bibi
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  4. Marcelo Fernández-Lahore
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Corresponding author

Correspondence to Marcelo Fernández-Lahore .

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Editors and Affiliations

  1. Leibniz-Institut für Oberflächenmodifizierung, Leipzig, Germany

    Senta Reichelt

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Singh, N.K., DSouza, R.N., Bibi, N.S., Fernández-Lahore, M. (2015). Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography. In: Reichelt, S. (eds) Affinity Chromatography. Methods in Molecular Biology, vol 1286. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2447-9_16

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  • DOI: https://doi.org/10.1007/978-1-4939-2447-9_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2446-2

  • Online ISBN: 978-1-4939-2447-9

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