Protein purification with vapor-phase carbon dioxide

Gaseous CO2 was used as an antisolvent to induce the fractional precipitati on of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and t heir mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added conti nuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure-depende nt volumetric expansion of DMSO and a consequent reduction in solvent stren gth of DMSO towards dissolved proteins. View cell experiments were conducte d to determine the pressures at which various proteins precipitate from DMS O. The solubility of each protein in CO2-expanded DMSO was different, illus trating the potential to separate and purify proteins using gaseous antisol vents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PA GE) was used to quantify the separation of lysozyme from ribonuclease, alka line phosphatase from insulin, and trypsin from catalase. Lysozyme biologic al activity assays were also performed to determine the composition of prec ipitates from DMSO initially containing lysozyme and ribonuclease. SDS-PAGE characterizations suggest that the composition and purity of solid-phase p recipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonucle ase, and trypsin precipitates recovered substantial amounts of biological a ctivity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor-phase antisolvents, which are easily s eparated and recovered from proteins and liquid solvents upon depressurizat ion, appear to be a reliable and effective means of selectively precipitati ng proteins. (C) 1999 John Wiley & Sons, Inc.