High-level expression and one-step purification of cyclic amidohydrolase family enzymes.
The cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells. With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures. Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification. The expression levels of the fusion proteins account for 20-35% of the total protein in E. coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes. The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes.
Kim
G J
GJ
Department of Molecular Science and Technology, Ajou University, San5, Woncheon-dong, Paldal-gu, Suwon, 442-749, Korea.
Lee
D E
DE
Kim
H S
HS
D018528:ATP-Binding Cassette Transporters; D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D029968:Escherichia coli Proteins; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000581:Amidohydrolases