Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent

Abstract

A continuous supermacroporous matrix has been developed allowing direct capture of enzyme from non-clarified crude cell homogenate at high flow-rates. The continuous supermacroporous matrix has been produced by radical co-polymerization of acrylamide, allyl glycidyl ether and N, N′-methylene-bis(acrylamide) which proceeds in aqueous solution of monomers frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix having interconnected pores of 10–100 μm size. Iminodiacetic acid covalently coupled to the cryogel is a rendering possibility for immobilized metal affinity chromatographic purification of recombinant His-tagged lactate dehydrogenase, (His) 6-LDH, originating from thermophilic bacterium Bacillus stearothermophilus, but expressed in Escherichia coli. The large pore size of the adsorbent makes it possible to process particulate-containing material without blocking the column. No preliminary filtration or centrifugation is needed before application of crude extract on the supermacroporous column. A total of 210 ml crude homogenate, 75 ml of it non-clarified, was processed on a single 5.0 ml supermacroporous column at flow speeds up to 12.5 ml/min without noticeable impairment of the column properties. Mechanically the cryogel adsorbent is very stable. The continuous matrix could easily be removed from the column, dried at 70 °C and kept in a dry state. After rehydration and reinsertion of the matrix into an empty column, (His) 6-LDH was purified as efficiently as on the newly prepared column. The procedure of manufacturing the supermacroporous continuous cryogel is technically simple. Starting materials and initiators are cheap and available and are simply mixed and frozen under specified conditions. Altogether these qualities reveal that the supermacroporous continuous cryogels is a very interesting alternative to existing methods of protein purification from particulate-containing crude extracts.