Direct Capture of His6-Tagged Proteins using Megaporous Cryogels
What is it about?
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.
Why is it important?
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. In the procedure described herein, megaporous cryogels are synthesized, grafted with epoxides, and subsequently functionalized with IDA. Cu(II)ions are then chelated onto the IDA-cryogel backbone to obtain IMAC functionality.
The following have contributed to this page: Professor Hector M. Fernandez Lahore and Dr Naveen Kumar Singh
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