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.
Downstream Bioprocessing Laboratory, School of Engineering and Science, Jacobs University, Campus Ring 1, Bremen, 28759, Germany.
Naveen Kumar
NK
Singh
Roy N
RN
DSouza
Noor Shad
NS
Bibi
Marcelo
M
Fernández-Lahore
D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D059985:Cryogels / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry*; D016062:Porosity; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification*
D059985:Cryogels; C471213:His-His-His-His-His-His; D007093:Imidazoles; D008024:Ligands; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper