Chisholm J W, Gebre A K, Parks J S
Department of Pathology, Section on Comparative Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 -1040, USA.
J Lipid Res. 1999 Aug;40(8):1512-9.
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.
卵磷脂胆固醇酰基转移酶（LCAT）是一种血浆酶，可催化磷脂的sn-2脂肪酸与胆固醇的酯化反应。为便于大量分离LCAT并辅助未来的结构-功能研究，在中国仓鼠卵巢细胞（CHO）中表达了含有羧基末端组氨酸标签（H6）的LCAT。使用10 mM丁酸钠增强转录并采用PFX-CHO无蛋白培养基，在72小时内实现了高水平的CHO-hLCATH6表达（约15 mg L⁻¹）。通过钴金属亲和层析分离得到了纯度约为96%的纯酶，活性回收率为82±26%。CHO-hLCATH6和CHO-hLCAT的比活性相同（分别为26±6和26±3 nmol CE形成/μg⁻¹ h⁻¹）。CHO-hLCATH6的酶活性在4℃下超过60天保持稳定。使用由1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）、胆固醇和载脂蛋白A-I（80:5:1）组成的重组高密度脂蛋白（rHDL）进行的底物饱和研究表明，CHO-hLCATH6、CHO-hLCAT和纯化的血浆LCAT在底物胆固醇约为2 μM时的表观K(m)几乎相同。我们得出结论，羧基末端组氨酸标签化的LCAT是血浆LCAT和CHO-hLCAT的合适替代物。
