出版社：American Society for Biochemistry and Molecular Biology
摘要：Lecithin:cholesterol acyltransferase (LCAT) is the plasma enzyme that catalyzes esterification of the sn -2 fatty acid of phospholipid to cholesterol. To facilitate the isolation 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 hamster ovary cells (CHO). A high level of CHO-hLCATH6 expression (≈15 mg L-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 ± 26%. CHO-hLCATH6 and CHO-hLCAT species had identical specific activities (26 ± 6 and 26 ± 3 nmol CE formed μg-1 h-1, respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4°C in excess of 60 days. Substrate 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 app Km for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at ≈2 μ 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.
