Expression and purification of Escherichia coli beta-glucuronidase.
A strong and constitutive expression vector of Escherichia coli beta-glucuronidase with the isocitrate dehydrogenase promoter has been developed for producing a large amount of recombinant protein. More than 95% pure enzyme was obtained by a four step purification procedure-ammonium sulfate precipitation, DEAE ion-exchange chromatography, Superose 12 gel filtration, and hydroxyapatite steric ion-exchange chromatography. The overexpressed gene can produce 23 mg of pure enzyme from one liter of bacterial culture.
Aich
S
S
Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada.
Delbaere
L T
LT
Chen
R
R
D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase; D005966:Glucuronidase