Winters M A, Frankel D Z, Debenedetti P G, Carey J, Devaney M, Przybycien T M
Department of Chemical Engineering, Princeton University, Princeton, New Jersey 08544-5263, USA.
Biotechnol Bioeng. 1999 Feb 5;62(3):247-58. doi: 10.1002/(sici)1097-0290(19990205)62:3<247::aid-bit1>3.0.co;2-s.
Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2-expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins.
气态二氧化碳被用作反溶剂，以诱导碱性磷酸酶、胰岛素、溶菌酶、核糖核酸酶、胰蛋白酶及其混合物从二甲基亚砜（DMSO）中分步沉淀。将压缩二氧化碳连续等温地添加到静止的DMSO溶液中（气态反溶剂，GAS）。二氧化碳的溶解伴随着DMSO明显的、与压力相关的体积膨胀，以及DMSO对溶解蛋白质的溶剂强度的相应降低。进行了视窗细胞实验，以确定各种蛋白质从DMSO中沉淀时的压力。每种蛋白质在二氧化碳膨胀的DMSO中的溶解度不同，这说明了使用气态反溶剂分离和纯化蛋白质的潜力。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）来量化溶菌酶与核糖核酸酶、碱性磷酸酶与胰岛素、胰蛋白酶与过氧化氢酶的分离。还进行了溶菌酶生物活性测定，以确定最初含有溶菌酶和核糖核酸酶的DMSO沉淀的组成。SDS-PAGE表征表明，通过反溶剂的压力可以精确控制从含有多种蛋白质的溶液中沉淀出的固相的组成和纯度。胰岛素、溶菌酶、核糖核酸酶和胰蛋白酶沉淀在重新溶解于水性介质后恢复了大量的生物活性。然而，碱性磷酸酶发生了不可逆的变性。气相反溶剂在减压后很容易从蛋白质和液体溶剂中分离和回收，似乎是选择性沉淀蛋白质的可靠而有效的手段。
