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 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.
Chisholm
J W
JW
Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 -1040, USA.
Gebre
A K
AK
Parks
J S
JS
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004790:Enzyme Induction; D015870:Gene Expression; D006639:Histidine; D006801:Humans; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D011994:Recombinant Proteins; D006639:Histidine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase