Preparation of a pH-sensitive affinity precipitation polymer and its application in purification of trypsin

Affinity precipitation is a potential protein purification technique, and preparation of affinity macroligands is a key problem. Until now, only a few polymers could be used for proteins purification in laboratory scale. In this paper, a reversibly water-soluble and pH-sensitive copolymer was synthesized by radical polymerization using methyl acrylic acid (MAA), methyl methacrylate (MMA), methacrylic acid 2-(dimethylamino) ethyl ester (DMAEMA) and N-hydroxymethyl acrylamide (NHMA). Its recovery was over 95% by adjusting pH to its isoelectric point (pI=6.5). The classical epichlorohydrine activation method was used to activate the hydroxyl groups on the copolymer, and then p-amino benzamidine was coupled to the activated copolymer as an affinity ligand for trypsin adsorption. Various affinity macroligands with different ligand densities were prepared. The polymer with ligand density of 39.5μmol/g, was used for purification of trpsin. The adsorption capacity of trypsin was found to be 132.5mg/g polymer. Trypin was purified from 267.2 to 1637.1IU/mg protein with 82.6% of activity yield and 6.13 folds of purification factor. The SDS-PAGE analysis demonstrated that trypsin with electrophoresis purity was obtained.