title: Purification and biophysical characterization of the core protease domain of anthrax lethal factor.
abstract: Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn(2+) binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF(672-776)) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ((1)H, (13)C, (15)N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn(2+), suitable for high resolution structural analysis by NMR.
authors.affiliation: Department of Pharmacy, University of Patras, GR-26504 Patras, Greece.
authors.forename: Petros V
authors.initials: PV
authors.lastname: Gkazonis
authors.forename: Georgios A
authors.initials: GA
authors.lastname: Dalkas
authors.forename: Christos T
authors.initials: CT
authors.lastname: Chasapis
authors.forename: Alexios
authors.initials: A
authors.lastname: Vlamis-Gardikas
authors.forename: Detlef
authors.initials: D
authors.lastname: Bentrop
authors.forename: Georgios A
authors.initials: GA
authors.lastname: Spyroulias
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D001408:Bacillus anthracis / Q000201:enzymology*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020134:Catalytic Domain; D019906:Nuclear Magnetic Resonance, Biomolecular; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin; D010447:Peptide Hydrolases