Smart Polymers and Protein Purification

Abstract

Smart polymers undergo fast and reversible changes in microstructure triggered by small changes of medium property (pH, temperature, ionic strength). These properties of smart polymers were exploited for the development of new protein purification techniques: affinity precipitation, affinity partitioning, and temperature-induced elution in dye-affinity chromatography. Different smart polymers were used for affinity precipitation: Eudragit S 100 (a copolymer of methacrylic acid and methyl methacrylate which precipitates on decreasing pH); stoichiomet-ric polyelectrolyte complexes of poly(methacrylate) and poly(N-ethyl-4-vinylpyridium bromide); poly(N-vinyl caprolactam), and poly(N-iso-propylacrylamide) (both precipitate on heating aqueous solutions to 35–40°C or increase in ionic strength). Recombinant thermostable lac-tate dehydrogenase from a thermophile Bacillus stear other mophilus was purified by affinity partitioning in an aqueous two-phase polymer system formed by dextran and a copolymer of N-vinyl caprolactam and 1-vinyl imidazole. The enzyme partitioned preferentially into the copolymer phase in the presence of Cu ions. The thermoprecipitation of the copolymer at 45°C resulted in an aqueous solution of the purified en- At elevated temperatures, poly-VCL molecules are in a compact globule conformation, capable of binding only to a few ligands on the matrix. The enzyme lactate dehydrogenase from porcine muscle has better access to the ligands and binds to the column. With a decrease in temperature, the polymer molecules undergo transition to a more expanded coil conformation. Polymer molecules interact now with more ligands and begin to compete with the bound enzyme for the ligands. Finally, the bound enzyme is replaced by the expanded polymer chains. This system was used for lactate dehydrogenase purification. Porcine muscle homogenate was applied on a column until breakthrough at 40°C. The foreign proteins were washed out with 0.1 M KC1; then the column was cooled to room temperature and virtually homogeneous lactate dehydrogenase was eluted with the same buffer. The purification factor was 17; recovery was 90% (13).