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

Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn -2 fatty acid of phospholipid to cholesterol. To facilitate the isola- tion of large quantities of LCAT and to assist in future structure-function studies, LCAT containing a carboxy- terminal histidine-tag (H6) was expressed in Chinese ham- ster ovary cells (CHO). A high level of CHO-hLCATH6 ex- pression ( < 15 mg L 2 1 ) was achieved over a 72-h period using 10 m M sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( < 96%) was isolated by cobalt metal affinity chromatography with an activity yield of 82 6 26%. CHO-hLCATH6 and CHO- hLCAT species had identical specific activities (26 6 6 and 26 6 3 nmol CE formed m g 2 1 h 2 1 , respectively). The enzy- matic activity of CHO-hLCATH6 was stable at 4 8 C in excess of 60 days. Substrate saturation studies, using rHDL com- posed of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC), cholesterol, and apolipoprotein A-I (80:5:1) indi- cated that the app K m for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at < 2 m M substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT. —Chisholm, J. W., A. K. Gebre, and J. S. Parks. Characterization of C-terminal histidine-tagged human recombinant lecithin:cholesterol acyltransferase. J. Lipid Res. 1999. 40: 1512-1519.