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 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® cloning system to generate the E. histolytica expression vector pAHDEST. 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-Myctagged, showed a cytoplasmic localization in transfected trophozoites. This expression vector with the Gateway® system should facilitate investigation into the functions of novel proteins in E. histolytica. Keywords Entamoeba histolytica; Gateway® Vector; EHI_144490Entamoeba histolytica is an enteric aerotolerant parasite that can colonize the large intestine, and invade through the intestinal epithelium to cause amebic colitis and liver abscess [1]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. fairly large data sets [2][3][4][5][6][7]. An efficient and high throughput method is needed for cloning and analyzing the function of the proteins encoded by these transcripts. NIH Public AccessThe Gateway® (Invitrogen) system has been used successfully to stably express open reading frames (ORFs) from P. falciparum by recombinational cloning, which like E. histolytica, contains an AT-rich genome [8][9][10]. The Gateway® system also has the potential to rapidly transfer cloned inserts from one vector to another [11]. To construct the E. histolytica 'Destination' plasmid we used the well-characterized plasmid pGir308 as the backbone of the new construct [12]. This vector uses the E. histolytica ferredoxin promoter, which drives strong expression of cloned genes in vivo and in vitro [2,13]. The vector also carries a hygromycin resistance gene, which permits the use of this vector in co-infection studies with other E. histolytica shuttle vectors that carry G418-selectable markers [14].The vector pGir308 was digested with Xb...

Aminoglycoside modifying enzymes

“…These enzymes mediate resistance to hygromycin and are not clinically relevant. These genes have been used in the construction of cloning vehicles for both prokaryotes and eukaryotes (Abhyankar et al, 2009; Gritz and Davies, 1983). …”

A Genome-Wide Over-Expression Screen Identifies Genes Involved in Phagocytosis in the Human Protozoan Parasite, Entamoeba histolytica

“…E. histolytica cells were transformed with a cDNA over-expression library constructed in the episomal pAH-DEST expression vector [23] . Expression from this vector is constitutively by sequences derived from the upstream region of the E. histolytica ferredoxin gene [24] .…”

“…The LR Clonase system (Invitrogen) was used to transfer cDNA inserts from pENTR™222 to pAH-DEST [23] , a Gateway-compatible E. histolytica expression vector (kind gift from C.A. Gilchrist and W.A.…”

Production of recombinant proteins from protozoan parasites

“…This system has been applied successfully to P. falciparum single-exon genes, with an 84% cloning efficiency and a 100% success rate when subcloned into three expression vectors [31]. The same approach was used to identify novel malaria antigens for DNA vaccines [86] and it is being adopted for cloning and expressing genes in Entamoeba histolytica [87]. Similarly, this system can be quickly adopted for any parasite of interest to build a battery of expression vectors designed specifically to express and produce recombinant proteins in any parasite specific heterologous system developed [88].…”