Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography
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 wordsProtein purification Graft-copolymerization Megaporous cryogels IMAC Affinity chromatography
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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