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

Protein Expr Purif. 2016 Sep:125:1-6. doi: 10.1016/j.pep.2015.09.003. Epub 2015 Sep 9.

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.

Keywords: BacMam; HEK293S GnTI−; Kifunensine; LCAT; Lecithin–cholesterol acyltransferase; Underglycosylated.

MeSH terms

  • Animals
  • Biological Transport
  • Cholesterol / metabolism
  • Enzyme Activation
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Lipoproteins, HDL / chemistry
  • Lipoproteins, HDL / metabolism
  • Phosphatidylcholine-Sterol O-Acyltransferase / chemistry
  • Phosphatidylcholine-Sterol O-Acyltransferase / genetics*
  • Phosphatidylcholine-Sterol O-Acyltransferase / isolation & purification
  • Phosphatidylcholine-Sterol O-Acyltransferase / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sf9 Cells

Substances

  • Lipoproteins, HDL
  • Recombinant Proteins
  • Cholesterol
  • Phosphatidylcholine-Sterol O-Acyltransferase