Expression, refolding and purification protocol for Trypanosomaevansi adenosine deaminase protein in e.coli rosetta gami

Trypanosomaevansi causes a highly pathogenic disease in equines popularly known as "Surra". In Brazilian Pantanal, outbreaks are recorded due to the large population of horses. Losses to livestock are due to inefficient treatments. Trypanosomes are vulnerable to purine metabolism because they do not have the De novopathway and they satisfy their requirements by salvaging the preformed bases demonstrating acomplete purine dependence on their hosts. Among the components of this system, we highlight Adenosine,which has its concentration controlled by the enzyme Adenosine Deaminase (ADA). The objectives of this study were amplifying, cloning and sequencingADAgene inTrypanosomaevansi (TeADA). The coding region of TeADAwas amplified from the T. evansi genomic DNA and the 1857 bp sequence showed a high degree of similarity (95%) with T. bruceiADA gene (TbADA). The amplicon was cloned in apGEM-T Easy® vector and expressed using pET30 vector. Protein expression wasanalysed usingSDS-PAGE and the best condition to obtain a protein of approximately 68 kDa was 18 °C for 24 hours and induction with 0.05 mM IPTG.Protein was solubilized through refolding and it was purified by affinity chromatography.Expression of TeADAwas confirmed by Western blot.