Development of the Gateway system for cloning and expressing genes in Entamoeba histolytica

Abstract

The early branching eukaryote Entamoeba histolytica is a human parasite that is the etiologic agent of amebic dysentery and liver abscess. The sequencing of the E. histolytica genome combined with the development of an E. histolytica microarray has resulted in the identification of several distinct gene expression profiles associated with virulence. The function of many modulated transcripts is unknown and their role in pathogenicity is unclear. They however represent a pool of potential virulence factors that could be targets for the development of novel therapeutics. Efficient tools and methods to characterize these novel virulence-associated genes and proteins would be beneficial. Here we report the use of the Gateway((R)) cloning system to generate the E. histolytica expression vector pAH-DEST. To test the usefulness of this system, the vector was used to construct a plasmid containing a recombinant version of the locus EHI_144490, which encoded a protein of unknown function. The recombinant gene was expressed and the recombinant protein, which was strep-myc-tagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway((R)) system should facilitate investigation into the functions of novel proteins in E. histolytica.

Fig. 1

(A) Construction of the E. histolytica Gateway® expression vector pAH-DEST. The tet^R cassette from pGIR308 was replaced with the Gateway® (Invitrogen cat. # 11828-029) cassette containing attR recombination sites flanking a ccdB gene and a chloramphenicol-resistance (cm^R) gene. The new expression vector contained an ampicillin^R gene for bacterial selection. (B) Affinity purification of recombinant EHI_144490. The cell lysate from trophozoites expressing Strep-Myc tagged recombinant protein was affinity-purified using a Strep-Tactin column (IBA, Gmbh) as per the manufacturer’s instructions and immunoblotted. The blot was probed with anti-Myc antibody. As expected a predominant 68kDa band was seen in whole cell lysate (lane 1) as well as in the purified fraction (lane 2). Minor higher molecular mass bands that might represent protein aggregation or post-translational modifications were also identified. No proteins recognized by the anti-Myc antibody were seen in lysate (lane 3) or purified fraction (lane 4) controls prepared from trophozoites transfected with pAH DEST vector alone.

Fig. 2

Cytoplasmic location of recombinant EHI_144490. HM1: IMSS trophozoites were transfected with the Gateway® construct expressing recombinant EHI_144490. Nuclei were stained with DAPI (blue, panel A). The recombinant protein was detected with monoclonal anti-Myc antibody and Cya 3-conjugated donkey anti-mouse antibody (panel B). The bright field image is shown in panel C, and panel D shows the merged images. The recombinant protein was cytoplasmic (red, panel B and D). Amebae probed with secondary antibody alone did not show staining (data not shown). The images were obtained using a Zeiss LSM 410 laser scanning confocal microscope.