Efficient expression, purification, and characterization of C-terminally tagged, recombinant human asparagine synthetase.
Several lines of evidence suggest that up-regulation of asparagine synthetase (AS) in human T-cells results in metabolic changes that underpin the appearance of asparaginase-resistant forms of acute lymphoblastic leukemia (ALL). Inhibitors of human AS therefore have potential as agents for treating leukemia and tools for investigating the cellular basis of AS expression and drug-resistance. A critical problem in developing and characterizing potent inhibitors has been a lack of routine access to sufficient quantities of purified, reproducibly active human AS. We now report an efficient protocol for preparing multi-milligram quantities of C-terminally tagged, wild type human AS in a baculovirus-based expression system. The recombinant enzyme is correctly processed and exhibits high catalytic activity. Not only do these studies offer the possibility for investigating the kinetic behavior of biochemically interesting mammalian AS mutants, but such ready access to large amounts of enzyme also represents a major step in the development and characterization of inhibitors that might have clinical utility in treating asparaginase-resistant ALL.
Ciustea
Mihai
M
Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
Gutierrez
Jemy A
JA
Abbatiello
Susan E
SE
Eyler
John R
JR
Richards
Nigel G J
NG
D000595:Amino Acid Sequence; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D001217:Aspartate-Ammonia Ligase