Cloning, Expression, and Characterization of the Aminoethylphosphonate Pathway in Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, is densely coated with virulence factors, mostly anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). The core of a GPI molecule is conserved among eukaryotes, containing the canonical EtNP-Man₃-GlcN-myo-Inositol-PO₄-lipid structure. T. cruzi is possibly the only eukaryote capable of utilizing either ethanolaminephosphate (EtNP) or aminoethylphosphonate (AEP) as the linker between proteins and the GPI-anchor. Besides the conversion of phosphoenolpyruvate (PEP) into 3-phosphonopyruvate, the T. cruzi’s biosynthetic pathway of AEP remains unsolved. Gene survey reveals two enzymes, namely, phosphonopyruvate decarboxylase (PPDC) and aminoethylphosphonate transamidase (AEPT). This study aims to clone, express, and characterize PPDC and AEPT, because their potential chemotherapeutic targets. Specific oligonucleotides were designed to amplify the open-reading frame of PPDC and AEPT genes. The amplicons were cloned into pGEM-T easy vector and sequenced. AEPT and PPDC genes are 1,203 and 1,359 bp in length encoding proteins of 400 and 452 amino acids, respectively. The inserts were sub-cloned into pET28a and pRSET-A expression vectors and transformed into E. coli BL-21 strain. The expression was induced for 3h at 37°C with 1 mM IPTG. SDS-PAGE and blotting analyses indicated both proteins were expressed in bacteria. Biochemical characterization of PPDC and AEPT is underway.