Affinity precipitation - an alternative to fluidized bed adsorption?

In affinity precipitation reversibly water-soluble affinity macroligands, i.e., polymers bearing an affinity tag, are used to first bind and than co-precipitate the target molecule. In this paper a new type of affinity macroligand is introduced. The molecules were constructed by group transfer polymerization of N,N-diethylacrylamide. They were small (less than 15 monomeric units per molecule) and very homogeneous in size (polydispersity of the molecular mass <1.2). Each base polymer started with carboxylic acid group to which site the affinity tag (the protease inhibitor m-aminophenylboric acid) was later bound by carbodiimid coupling (yield >50%). An inhibition constant, K-1, of less than 10 mu mol/l was determined for the final affinity macroligand. Low temperature and high salt concentrations were shown to be beneficial to binding. Successful affinity precipitation of the serin protease Substilisin carlsberg required a further lowering of the affinity constant by at least one-order of magnitude, which was accomplished by the addition of 5% (v/v) of ethylene glycol. The ethylene glycol effect was assumed to be due to the formation of a cyclic ester with the phenylboric acid of the affinity ligand. (C) 1998 Elsevier Science B.V. All rights reserved.