Expression, purification, crystallization and structure solution of glyceraldehyde-3-phosphate dehydrogenase from the...

Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks, causing a global cattle disease. As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance, glycolytic enzymes are possible targets for drug development against Babesia. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) has been one of such targets, against several parasitic organisms. It performs the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate in the presence of nicotinamide adenine dinucleotide. The protocol for Babesia bovis Glyceraldehyde-3-Phosphate Dehydrogenase (BbGAPDH) expression and purification has been developed to yields of 28 mg of pure protein per liter of culture medium, with a specific activity of 55.5 ± 6.99 U mg^-1 after His-tag removal, indicating preservation of enzymatic activity. For crystallization, the His-tag removal proved essential. Crystals diffracted to 3.12 Å resolution in the P3₁21 space group; NAD⁺ cofactor molecules are observed in their respective sites. Comparisons to the theoretical model indicate a number of side chain conformation differences. These results provide support for future enzyme inhibition assays, in addition to crystallization assays with potential inhibitors.