Purification of recombinant lecithin: cholesterol acyltransferase.
Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned 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 revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.
Nair
M P
MP
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, USA.
Kudchodkar
B J
BJ
Pritchard
P H
PH
Lacko
A G
AG
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D006801:Humans; D007668:Kidney; D008647:Mesocricetus; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D011993:Recombinant Fusion Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase