Purification and properties of human glucose-6-phosphate dehydrogenase made in E. coli.
The cDNA for the X-chromosome encoded human glucose-6-phosphate dehydrogenase (G6PD) has been expressed in E. coli and the enzyme purified to homogeneity, using a simple one-step fractionation on 2'5'-ADP-Sepharose. By selecting one of several different expression vectors and by optimizing culture conditions a yield of more than 10 mg of pure enzyme per liter of culture is obtained reproducibly. When the recombinant enzyme and authentic G6PD purified from normal human red cells were compared, they proved to be indistinguishable by the following criteria: electrophoretic mobility in both native and denaturing conditions, the Km values for glucose 6-phosphate and NADP and the Ki value for NADPH. The recombinant enzyme, unlike the red cell enzyme, retained 100% activity when stored at 4 degrees C for over 1 year.
Bautista
J M
JM
Department of Haematology, Royal Postgraduate Medical School, London, U.K.
Mason
P J
PJ
Luzzatto
L
L
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014960:X Chromosome
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D005954:Glucosephosphate Dehydrogenase