Glycosomal glyceraldehyde-3-phosphate dehydrogenase of Trypanosoma brucei and Trypanosoma cruzi: expression in Escherichia coli, purification, and characterization of the enzymes.
The Trypanosoma brucei and Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenases have been overexpressed in Escherichia coli using a T7 expression system. These enzymes have been highly purified by ammonium sulfate precipitation, followed by phenyl-Sepharose and phospho-ultrogel chromatography. From 1 liter of bacterial culture, we obtained 4.4 mg of T. brucei enzyme, with a specific activity of 147 units/mg, and 26.6 mg of T. cruzi enzyme, with a specific activity of 122 units/mg. Both proteins have a similar subunit mass of 38 kDa. Some physicochemical and kinetic properties have been determined and compared with those reported for the authentic T. brucei enzyme. The two enzymes appear to be very similar, except for the dependence of their activity on ionic strength.
Hannaert
V
V
Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Opperdoes
F R
FR
Michels
P A
PA
D000818:Animals; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D017125:Genes, Protozoan / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008830:Microbodies / Q000201:enzymology*; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases