Use of the avidin (imino)biotin system as a general approach to affinity precipitation

Affinity precipitation, especially secondary effect affinity precipitation, has repeatedly been suggested as a valuable technique for the biotechnical downstream process. The present lack of applications is related to the sca rcity of predictable affinity macroligands and to the fact that rather high affinity constants are required in affinity precipitation (K-D < 10(-10)). The latter are rarely found in nature, at least in the case of small affin ity ligands (affinity tags), and are usually difficult to handle (complex d issociation) once one has found them. In this article we describe a new typ e of thermoresponsive affinity macroligand. The base polymer (poly-N-isopro pylacrylamide, or PNIPAAm) is produced by chain transfer polymerization. As a consequence, the structure, as well as the solubility behavior, is very homogeneous (polydispersity < 2.2), whereas the average molecular mass is s mall (<5000 g/mol). In pure water, the base polymer shows sharp thermopreci pitation at 32.2<degrees>C. Each oligomer carries a single amino end group, which allows easy and defined coupling of the affinity ligand, while prese rving the ligand's activity to the highest possible degree. Herein, the oli gomer was coupled to iminobiotin. The ensuing affinity macroligand has a hi gh affinity to avidin (and avidin-tagged molecules) at elevated pH (<10), b ut releases the avidin easily at lower pH (<approximate to>4). The affinity macroligands were used to purify avidin from solutions containing large am ounts of lysozyme as well as from cell culture supernatants containing 5% f etal calf serum. In both cases, pure avidin was recovered (residual protein contamination below the detection limit), with yields of >90%. (C) 2001 Jo hn Wiley & Sons, inc.