title: Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate.
abstract: This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Mirica
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Lockett
authors.forename: Phillip W
authors.initials: PW
authors.lastname: Snyder
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Shapiro
authors.forename: Eric T
authors.initials: ET
authors.lastname: Mack
authors.forename: Sarah
authors.initials: S
authors.lastname: Nam
authors.forename: George M
authors.initials: GM
authors.lastname: Whitesides
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D005590:Fractional Precipitation / Q000379:methods*; D006801:Humans; D008024:Ligands; D012995:Solubility; D013449:Sulfonamides / Q000737:chemistry*
chemical_list: D008024:Ligands; D013449:Sulfonamides; D002256:Carbonic Anhydrases; D000645:Ammonium Sulfate