Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica

Abstract

Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of ∼500 μg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D 1H– 15N HSQC spectrum of a uniformly 13C/ 15N-labeled sample was achieved using a combinatorial selective 15N-labeling approach coupled with available 1H N chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D 1H– 15N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.