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

• Published in: Journal of lipid research Vol. 40; no. 8; pp. 1512 - 1519
• Main Authors: Chisholm, J W, Gebre, A K, Parks, J S
• Format: Journal Article
• Language: English
• Published: United States Elsevier 01-08-1999
• Subjects: Animals; butyric acid; CHO Cells; Cloning, Molecular; cobalt metal affinity chromatography; Cricetinae; Enzyme Induction; Gene Expression; his-tag; Histidine; Humans; LCAT; phosphatidylcholine-O-cholesterol acyltransferase; Phosphatidylcholine-Sterol O-Acyltransferase - biosynthesis; Phosphatidylcholine-Sterol O-Acyltransferase - genetics; Phosphatidylcholine-Sterol O-Acyltransferase - isolation & purification; Protein Engineering; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification
• ISSN: 0022-2275
• DOI: 10.1016/s0022-2275(20)33395-2

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 ( approximately 15 mg L(-1)) was achieved over a 72-h period using 10 mm sodium butyrate to enhance transcription and PFX-CHO protein-free medium. The pure enzyme ( approximately 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 microg(-1) h(-1), respectively). The enzymatic activity of CHO-hLCATH6 was stable at 4 degrees 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 appK(m) for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at approximately 2 microm substrate cholesterol. We conclude that carboxy-terminal histidine-tagged LCAT is a suitable replacement for both plasma LCAT and CHO-hLCAT.