Expression, purification, and characterization of histidine-tagged mouse monoglyceride lipase from baculovirus-infected insect cells.
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.
Karlsson
M
M
Section for Molecular Signalling, Lund University, Lund, S-221 00, Sweden.
Tornqvist
H
H
Holm
C
C
D000273:Adipose Tissue / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005989:Glycerides / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000821:virology
D005989:Glycerides; D011994:Recombinant Proteins; D006639:Histidine; C005953:monoolein; D008994:Monoacylglycerol Lipases