Producing a recombinant flavin-containing monooxygenase from Coffea arabica in Escherichia coli for screening of potential natural substrates.
Only few biological functions have been related with flavin-containing monooxygenases (FMOs) in plants, such as specific roles in auxin biosynthesis, pathogen defense, and metabolism of glucosinolates. Biochemical characterization using recombinant proteins is a promising approach to determine the precise specificity of plant FMOs for potential natural substrates. FMOs may be very difficult to express in a soluble form due to their highly hydrophobic nature and this can be improved by fusing them to solubility-enhancing proteins, such as maltose-binding protein (MBP) and N-utilization substance A (NusA). Here we describe the expression of a recombinant FMO from Coffea arabica as a maltose-binding protein fusion in Escherichia coli and its purification by affinity chromatography, producing a ready-to-use protein for enzymatic activity assays.
Cesarino
Igor
I
Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, SP, Brazil.
Mazzafera
Paulo
P
D001688:Biological Products / Q000378:metabolism*; D002846:Chromatography, Affinity; D040503:Coffea / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D005818:Genetic Engineering / Q000379:methods*; D010105:Oxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001688:Biological Products; D011993:Recombinant Fusion Proteins; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)