Simultaneous Purification and Refolding of Proteins by Affinity Precipitation and Macro (Affinity Ligand)-Facilitated Three Phase Partitioning (MLFTPP)

Abstract

This chapter describes two simple interrelated non-chromatographic methods of protein purification. In the first method, called affinity precipitation, inherent affinity of reversibly soluble–insoluble polymers (also called stimuli-sensitive or smart polymers) is exploited to form an affinity complex in free solution with target protein. The affinity complex is precipitated by a suitable change in the medium. The desired protein is dissociated from the smart polymer. In the second method called macro (affinity ligand)-facilitated three phase partitioning (MLFTPP), the affinity complex is precipitated at an interface between upper t-butanol-rich phase and lower aqueous phase. These three phases are achieved by adding appropriate amounts of ammonium sulfate and t-butanol to the initial crude extract. In the first protocol, sequential MLFTPP is used with two different smart polymers to purify pectinase and cellulase from a single crude preparation. The second protocol illustrates the application of the affinity precipitation in simultaneous purification and refolding of a urea-denatured xylanase.