Purification and characteristics of recombinant human folylpoly-gamma-glutamate synthetase expressed at high levels in insect cells.
Folylpoly-gamma-glutamate synthetase activity is central to the operation of folate metabolism and is essential for the survival of mammalian stem cell populations but the very low levels of endogenous expression of this enzyme have greatly limited its study. We now report the expression of cytosolic folylpoly-gamma-glutamate synthetase (FPGS) cloned from human leukemic cells in baculovirus-infected insect cells at levels of 4-5% of the total soluble protein of the cells. As was the case with endogenously expressed mammalian FPGS, recombinant enzyme was quantitatively blocked at the amino terminus in spite of the large-scale production in insect cells. A three-step purification procedure resulted in an overall yield of 7-35 mg per liter of culture with a recovery of about 50% and purity approximately 95%; pure enzyme was stable to storage for extended periods. Pure protein had a specific activity of 25 micromol h(-1)mg(-1) with aminopterin as a substrate and used a broad spectrum of folates as substrates. The pure enzyme also carried out ATP hydrolysis in the absence of a folate substrate or glutamic acid; this partial reaction occurred at a k(cat) about 0.4% that of the full reaction. In vitro, this single protein added several (1-8) moles of glutamic acid per mole of folate analog, the same spectrum of folate polyglutamates as seen in vivo. The quantities of pure enzyme achievable in insect cells should allow functional and structural studies on this enzyme.
Sanghani
P C
PC
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California, 90033, USA.
Moran
R G
RG
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011624:Pteroylpolyglutamic Acids / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity
D011624:Pteroylpolyglutamic Acids; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase