title: 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.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Punjab, India.
authors.forename: Ipsita
authors.initials: I
authors.lastname: Roy
authors.forename: Kalyani
authors.initials: K
authors.lastname: Mondal
authors.forename: Munishwar N
authors.initials: MN
authors.lastname: Gupta
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D008024:Ligands; D011506:Proteins