title: Production in Escherichia coli, folding, purification and characterization of notexin with wild type sequence and with N-terminal and catalytic site mutations.
abstract: Notexin (Ntx) is a group I phospholipase A2 (PLA2) protein, main component of the Australian snake Notechis scutatus scutatus venom. It is both a presynaptic neurotoxin and a myotoxin. In this work, for the first time, a method for the production and folding of recombinant Ntx was developed. Ntx was produced with wild type sequence (rNtx), with an extra peptide (T7-Ntx) or a methionine (M-Ntx) before Asn-1, and with Asn-1 substituted by alanine (Ntx-A1) or by serine (Ntx-S1). The proteins were analyzed for their catalytic and toxic activities. rNtx activity resulted to be comparable to that of the venom extracted protein. The Ntx N-terminus was found to have a major influence on both the catalytic and toxic activities of the protein. The first amino acid of snake venom PLA2s is highly conserved: it is an asparagine in about all group I PLA2s, while in most (>70%) of group II PLA2s it is a serine or an asparagine. Interestingly, Ntx-S1 resulted to be, for both enzymatic and toxic activities, the mutant most similar to the wild type protein. The role of the catalytic activity of Ntx in its toxicity was investigated by replacing the aspartic acid 49, involved in the coordination of the cofactor calcium ion, by a lysine. The obtained mutant (Ntx-K49) is deprived of catalytic activity but possesses a residual toxicity.
authors.affiliation: Istituto di Neuroscienze, CNR, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Morena
authors.initials: M
authors.lastname: Simonato
authors.affiliation: Dipartimento di Scienze Biomediche, Università degli Studi di Padova, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Morbiato
authors.affiliation: Dipartimento di Scienze Biomediche, Università degli Studi di Padova, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Zorzi
authors.affiliation: Dipartimento di Scienze Biomediche, Università degli Studi di Padova, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Caccin
authors.affiliation: Dipartimento di Scienze Biomediche, Università degli Studi di Padova, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Julián
authors.initials: J
authors.lastname: Fernández
authors.affiliation: Istituto di Neuroscienze, CNR, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Massimino
authors.affiliation: Centro Ricerche Interdipartimentale Biotecnologie Innovative, Università degli studi di Padova, Viale G. Colombo 3, 35131, Padova, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Polverino de Laureto
authors.affiliation: Istituto di Neuroscienze, CNR, Viale G. Colombo 3, 35131, Padova, Italy. Electronic address: fiorella.tonello@cnr.it.
authors.forename: Fiorella
authors.initials: F
authors.lastname: Tonello
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D004546:Elapid Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D054467:Phospholipases A2 / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D004546:Elapid Venoms; D011994:Recombinant Proteins; C005082:notexin; D054467:Phospholipases A2
keywords: Myotoxin; Neurotoxin; Notexin; Phospholipase A2; Protein folding; Snake venom