Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica.
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 approximately 500 microg/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.
Xun
Yang
Y
Department of Chemistry and School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand.
Tremouilhac
Pierre
P
Carraher
Colm
C
Gelhaus
Christoph
C
Ozawa
Kiyoshi
K
Otting
Gottfried
G
Dixon
Nicholas E
NE
Leippe
Matthias
M
Grötzinger
Joachim
J
Dingley
Andrew J
AJ
Kralicek
Andrew V
AV
D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D007473:Ion Channels; D009587:Nitrogen Isotopes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C041670:amoebapore proteins, protozoan