EFFECTIVE PURIFICATION METHOD OF LARGE MOLECULAR-WEIGHT PROTEINS USING CONVENTIONAL AOT REVERSE MICELLES

Hydrophilic bulky proteins with molecular weights larger than 60 kDa, such as catalase, beta-galactosidase, BSA and hemoglobin, can be easil y solubilized into a micro water pool of AOT reverse micelles by the i njection method. Those proteins and enzymes solubilized into reverse m icelles maintain their activities and native structures, and can be ba ck-extracted effectively to a new aqueous phase when the system pH is kept higher than their isoelectric points, pi, with low salt concentra tion, i.e. high water content. The back-extraction of hemoglobin is al so partially achieved under these conditions. The efficiency of back-e xtraction is strongly influenced by the pH values in the feed protein solution and in the aqueous solution used for back-extraction, as well as KCI concentration in the solution. The pH values of both solutions should be higher than the protein pI together with high Wo or low sal t concentration to preserve native structures and activities, since mo nomers and oligomers with relatively large molecular weight are likely to be denatured by association with ionic surfactant mediated by salt . In this case, both steric and electrostatic interactions between bul ky proteins and micelles play dominant roles in the separation. By red ucing interactions with micelles or surfactants, proteins solubilized in micelles can be effectively stabilized, and easily back-extracted t o the aqueous phase without inactivation. The present method suggests an another way for the effective bioseparation of bulky proteins with high yields of activity.