PURIFICATION OF RECOMBINANT LECITHIN - CHOLESTEROL ACYLTRANSFERASE

Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, h as been improved by Limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinan t BHK cells were grown until nearly confluent on multilayered flasks i n a fetal-calf-serum-enriched medium. Subsequently, the cells were was hed and supplied with serum free medium for 24-h periods, The conditio ned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step rev ealed the presence of a low-molecular-weight contaminant that exhibite d a carbohydrate/protein composition similar to proteoglycans. The maj or purified component contained LCAT activity and was homogeneous by a cryl-amide gel electrophoresis. (C) 1997 Academic Press.