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

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• 作者：Yang Xun ; Pierre Tremouilhac ; Colm Carraher ; Christoph Gelhaus ; Kiyoshi Ozawa ; Gottfried Otting ; Nicholas E. Dixon ; Matthias Leippe ; Joachim Grö ; tzinger ; Andrew J. Dingley ; Andrew V. Kralicek
• 关键词：Amoebapore A ; Antimicrobial proteins ; Cell-free synthesis ; NMR ; Pore formation ; Combinatorial isotope labeling
• 刊名：Protein Expression and Purification
• 出版年：2009
• 出版时间：November 2009
• 年：2009
• 卷：68
• 期：1
• 页码：22-27
• 全文大小：579 K

文摘

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.