Selective Precipitation and Purification of Monovalent Proteins Using Oligovalent Ligands and Ammonium Sulfate

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• 作者：Katherine A. Mirica ; Matthew R. Lockett ; Phillip W. Snyder ; Nathan D. Shapiro ; Eric T. Mack ; Sarah Nam ; George M. Whitesides
• 刊名：Bioconjugate Chemistry
• 出版年：2012
• 出版时间：February 15, 2012
• 年：2012
• 卷：23
• 期：2
• 页码：293-299
• 全文大小：380K
• 年卷期：v.23,no.2(February 15, 2012)
• ISSN：1520-4812

文摘

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鈥搇igand 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鈥搇igand 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鈥攆or which appropriate oligovalent ligands can be synthesized鈥攁nd combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.