Preparative reversed-phase liquid chromatography of proteins from rabbit skeletal troponin, a multi-protein complex

Abstract

A reversed-phase high-performance liquid chromatography protocol for purification of all proteins in a multi-protein (TnI, TnC, TnT, tropomyosin) complex from rabbit skeletal muscle has been developed, enabling efficient purification of sample amounts ranging from 43 mg of protein complex on a standard analytical column, to 1400 mg on a column of 21.2 mm I.D. and finally, to 5700 mg on a column of 50 mm I.D. Due to problems associated with scale-up procedures for these proteins (e.g. aggregation and/or solubility issues), an initial sample fractionation was devised whereby 50% of the TnC component was precipitated with acetonitrile prior to sample introduction on the RPLC column. By subsequently taking advantage of sample overload conditions to enhance the displacement effect between sample components, coupled with very slow gradient conditions (0.1% acetonitrile/min), we were able to achieve excellent protein separations at high yields of purified proteins.