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The Journal of Lipid Research, Vol. 40, 1512-1519, August 1999
Copyright © 1999 by Lipid Research, Inc.


Original Article

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

Jeffrey W. Chisholma, Abraham K. Gebrea, and John S. Parksa
a Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 -1040

Correspondence to: John S. Parks

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 ({approx}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 ({approx}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 appKm for CHO-hLCATH6, CHO-hLCAT, and purified plasma LCAT were nearly identical at {approx}2 µM 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.

Supplementary key words: phosphatidylcholine-O-cholesterol acyltransferase, LCAT, CHO cells, cobalt metal affinity chromatography, butyric acid, his-tag, glycosylation analysis, PFX-CHO


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