Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

Mem Inst Oswaldo Cruz. 2014 Dec;109(8):1081-5. doi: 10.1590/0074-0276140238. Epub 2014 Nov 21.

Abstract

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).

Publication types

  • Validation Study

MeSH terms

  • Blotting, Western
  • Expressed Sequence Tags / metabolism
  • Gene Expression / genetics*
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins
  • Life Cycle Stages / genetics
  • Mutagenesis, Insertional
  • Plasmids*
  • Restriction Mapping / methods*
  • Tetracycline / pharmacology
  • Trypanosoma cruzi / drug effects
  • Trypanosoma cruzi / genetics*

Substances

  • Green Fluorescent Proteins
  • Tetracycline