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.
Copyright 2000 Academic Press.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adipose Tissue / chemistry
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Animals
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Baculoviridae / genetics
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Cell Line
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Chromatography, Affinity
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Cloning, Molecular
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Electrophoresis, Polyacrylamide Gel
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Genetic Vectors
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Glycerides / chemistry
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Histidine / chemistry*
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Mass Spectrometry
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Mice
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Molecular Sequence Data
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Monoacylglycerol Lipases / chemistry
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Monoacylglycerol Lipases / isolation & purification*
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Monoacylglycerol Lipases / metabolism
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Rats
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Spodoptera / cytology
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Spodoptera / genetics
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Spodoptera / virology
Substances
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Glycerides
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Recombinant Proteins
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Histidine
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monoolein
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Monoacylglycerol Lipases