BacMam production of active recombinant lecithin–cholesterol acyltransferase: Expression, purification and characterization

• Lecithin cholesterol acyl transferase (LCAT) is important for cholesterol metabolism.
• The expression of LCAT by the BacMam system, purification and assay for activity.
• The use of kifunensine or HEK 293 GNTI− for the production of underglycosylated LCAT.
• Deglycosylated LCAT retains activity and is useful for structure determination.

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.