Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-avera ge molecular mass of 11 700 g/mol prepared by free radical polymerisation a t a 9:1 monomer molar ratio was separated into several fractions by IMAC an d thermal precipitation. The fraction with an average VI content of 8.5% wa s most efficient as a reusable displacer For IMAC of ovalbumin, lysozyme an d other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibite d a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depen ding on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quanti tatively from the protein fractions by thermal precipitation at 48 degreesC . Co-precipitation of lysozyme with the displacer was minimal in the presen ce of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the s upernatant was completely retained. Addition of free imidazole to the mobil e phase increased the rate of protein desorption and allowed better separat ion of egg white proteins and the displacer in the course of chromatography . The displacement profile of the egg white extract consisted of three zone s with different distributions of individual proteins characterised by SDS- PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0 .15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal pr ecipitation at 48 degreesC and subsequent dialysis against dilute hydrochlo ric acid (pH 2.5). (C) 2001 Elsevier Science B.V. All rights reserved.