Expression and purification of T. brucei APRT1 and APRT2 in E. coli

The T. brucei aprt1 and aprt2 genes were PCR amplified using oligonucleotides listed in Supplementary File ^S1, digested with BglII or BamHI and XhoI enzymes and ligated into the pSKB3 expression vector. The pSKB3 vector carries a 6 × His-tag and an AcTEV cleavage site upstream of BamHI restriction site. A BglII or BamHI restriction site was introduced at the 5′ end of the respective genes instead of the initiation methionine codon to maintain the 6 × His-tag and AcTEV recognition motif in frame with the genes. The verified plasmids were transformed into the BL21(DE3) E. coli cells. The 6xhis-tagged recombinant APRT1 protein was purified under native conditions using the ÄKTA prime plus instrument⁶. Elution fractions were analyzed by SDS-PAGE, and those containing APRT1 were pooled and dialyzed against 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol. Bradford protein assay (BioRad) was used to determine protein concentrations. The APRT1 recombinant protein was sent for antibody production to Davids Biotechnology (Regensburg, Germany). Recombinant APRT1 was subsequently used in all in vitro assays.