Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

ALONSO VL; RITAGLIATI C; CRIBB P; SERRA EC

MEMóRIAS DO INSTITUTO OSWALDO CRUZ.

FUNDACO OSWALDO CRUZ

Lugar: Rio de Janeiro; Año: 2014 vol. 109 p. 1081 - 1085

0074-0276

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).