Characterization of C-terminal histidine-tagged human recombinant lecithin: cholesterol acyltransferase

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catal yzes esterification of the sn-2 fatty acid of phospholipid to cholesterol, To facilitate the isolation of large quantities of LCAT and to assist in fu ture structure-function studies, LCAT containing a carboxyterminal histidin e-tag (H6) was expressed in Chinese hamster ovary cells (CHO), A high level of CHO-hLCATH6 expression (approximate to 15 mg L-1) was achieved over a 7 2-h period using 10 mM sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme (approximate to 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 +/- 26%, CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 +/- 6 and 26 +/- 3 nmol CE formed mu g(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees C in excess of 60 days. Su bstrate saturation studies, using rHDL composed of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5: 1) indicated that the appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plas ma LCAT were nearly identical at approximate to 2 mu M substrate cholestero l. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable re placement for both plasma LCAT and CHO-hLCAT.