Stimulus-Responsive Polymers for Bioseparation

Abstract

Affinity precipitation uses reversibly water-soluble affinity macroligands, i.e. stimulus-responsive polymers bearing one or several affinity ligands, to first capture and then co-precipitate a target molecule. Conventional, polymeric AML tend to suffer from a pronounced heterogeneity in both the structure of the polymer backbone and the affinity constants. In this paper a novel type of homogeneous, oligomeric AML is proposed, which carries one affinity ligand per AML in terminal position. The homogeneity of the AML translates into a very uniform precipitation behavior. Oligomeric AML-precursors prepared by chain or group transfer polymerization show a solubility, which is very similar to that reported previously for polymeric molecules of the same chemistry. Oligomers prepared by anionic polymerization are predominantly isotactic and show some deviations from this behavior. The affinity ligand is coupled to the oligomers via a reactive end group (e.g. an amino or carboxylic acid group) created during oligomer synthesis. An iminobiotin activated AML-precursor is used to recover avidin from a cell culture supernatant containing 5% FCS. Over 90% of the avidin are recovered in nearly pure form (residual protein contamination below the detection limit). This is one of the first purifications of a protein other than an enzyme by affinity precipitation with high yields. In a second example, the AML-precursor is activated by a single stranded DNA oligomer tag ((CTT)₇) and used to purify double stranded DNA molecules by triple helix affinity precipitation.