Destabilization domain approach adapted for regulated protein expression in the protozoan parasite Entamoeba histolytica

作者：Liu Yuk Chien; Singh Upinder^*

来源：International Journal for Parasitology, 2014, 44(10): 729-735.

DOI：10.1016/j.ijpara.2014.05.002

• Destabilized protein
• Enteric pathogen
• Amebiasis
• Protein degradation
• Inducible expression

A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica, however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddEKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddEKBP were rapid, with stabilization and degradation within 3 h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3 h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24 h after compound removal). Furthermore, we demonstrated that for the ddEKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica. Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen.

• 出版日期2014-9