Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells

Protein Expr Purif. 2000 Apr;18(3):286-92. doi: 10.1006/prep.1999.1194.

Abstract

Monoglyceride lipase (MGL) has been produced with the baculovirus-insect cell system. The mouse MGL cDNA was subcloned into a baculovirus transfer vector in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to apparent homogeneity was obtained by nickel-chelating chromatography. The final yield was 3 mg of pure enzymatically active MGL per liter of Sf9 cell suspension culture. Analysis by SDS-PAGE and mass spectrometry showed that the recombinant histidine-tagged enzyme had the expected molecular mass. With monoolein as substrate, the specific activity and the apparent K(m) were close to those of rat MGL of adipose tissue.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipose Tissue / chemistry
  • Animals
  • Baculoviridae / genetics
  • Cell Line
  • Chromatography, Affinity
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Vectors
  • Glycerides / chemistry
  • Histidine / chemistry*
  • Mass Spectrometry
  • Mice
  • Molecular Sequence Data
  • Monoacylglycerol Lipases / chemistry
  • Monoacylglycerol Lipases / isolation & purification*
  • Monoacylglycerol Lipases / metabolism
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spodoptera / cytology
  • Spodoptera / genetics
  • Spodoptera / virology

Substances

  • Glycerides
  • Recombinant Proteins
  • Histidine
  • monoolein
  • Monoacylglycerol Lipases

Associated data

  • GENBANK/AJ001118