High yield purification and first structural characterization of the full-length bacterial toxin CNF1(108 visite) Colarusso A, Caterino M, Fabbri A, Fiorentini C, Vergara A, Sica F, Parrilli E, Tutino ML
Biotechnol Prog (ISSN: 1520-6033electronic, 1520-6033linking), 2017 Oct 24; N/D: N/D-N/D.
Tipo di articolo: Journal Article,
Impact factor: 0, Impact factor a 5 anni: 0
Url: Non disponibile.
Parole chiave: Cnf1, Circular Dichroism, Protein Purification, Recombinant Protein Production, Secondary Structure Analysis,
Affiliazioni:
*** IBB - CNR *** Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant'Angelo, Via Cinthia, Napoli, 80126, Italy., Italian Center for Global Health, Istituto Superiore di Sanita, Viale Regina Elena, 299, Roma, 00161, Italy., CEINGE Biotecnologie Avanzate scarl, Via G. Salvatore, Napoli, 80100, Italy., Institute of Biostructures and Biomaging, CNR, Napoli, Italia Via Mezzocannone 16, Napoli, 80134, Italy.,
Riferimenti:
Non disponibile.
The Cytotoxic Necrotizing Factor 1 (CNF1) is a bacterial toxin secreted by certain Escherichia coli strains causing severe pathologies, making it a protein of pivotal interest in toxicology. In parallel, the CNF1 capability to influence important neuronal processes, like neuronal arborization, astrocytic support, and efficient ATP production, has been efficiently used in the treatment of neurological diseases, making it a promising candidate for therapy. Nonetheless, there are still some unsolved issues about the CNF1 mechanism of action and structuration probably caused by the difficulty to achieve sufficient amounts of the full-length protein for further studies. Here, we propose an efficient strategy for the production and purification of this toxin as a his-tagged recombinant protein from E. coli extracts (CNF1-H8). CNF1-H8 was expressed at the low temperature of 15 degrees C to diminish its characteristic degradation. Then, its purification was achieved using an immobilized metal affinity chromatography (IMAC) and a size exclusion chromatography so as to collect up to 8 mg of protein per liter of culture in a highly pure form. Routine dynamic light scattering (DLS) experiments showed that the recombinant protein preparations were homogeneous and preserved this state for a long time. Furthermore, CNF1-H8 functionality was confirmed by testing its activity on purified RhoA and on HEp-2 cultured cells. Finally, a first structural characterization of the full-length toxin in terms of secondary structure and thermal stability was performed by circular dichroism (CD). These studies demonstrate that our system can be used to produce high quantities of pure recombinant protein for a detailed structural analysis. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017.
248 Records (243 escludendo Abstract e Conferenze). Impact factor totale: 922.591 (900.298 escludendo Abstract e Conferenze). Impact factor a 5 anni totale: 958.218 (934.592 escludendo Abstract e Conferenze).
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