title: BacMam production of active recombinant lecithin-cholesterol acyltransferase: Expression, purification and characterization.
abstract: Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.
authors.affiliation: Protein Technologies, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States. Electronic address: wromanow@amgen.com.
authors.forename: William G
authors.initials: WG
authors.lastname: Romanow
authors.affiliation: Structural Biology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
authors.forename: Derek E
authors.initials: DE
authors.lastname: Piper
authors.affiliation: Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
authors.forename: Preston
authors.initials: P
authors.lastname: Fordstrom
authors.affiliation: Protein Technologies, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Thibault
authors.affiliation: Metabolic Disorders, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
authors.forename: Mingyue
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Structural Biology, Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.
authors.forename: Nigel P C
authors.initials: NP
authors.lastname: Walker
mesh_terms: D000818:Animals; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells
chemical_list: D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase
keywords: BacMam; HEK293S GnTI−; Kifunensine; LCAT; Lecithin–cholesterol acyltransferase; Underglycosylated