Use of a phosphopeptide as a ligand to purify phospholipase A 2 from the venom of Crotalus durisuss terrificus by affinity chromatography.

Autor: Saavedra SL; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina., Acosta G; Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain; CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain., Ávila L; Instituto Nacional de Producción de Biológicos, ANLIS Malbrán, Av. Vélez Sársfield 563, 1282 Buenos Aires, Argentina., Giudicessi SL; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina., Camperi SA; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina., Albericio F; Department of Organic Chemistry, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain; CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, University of Barcelona, 08028 Barcelona, Spain; School of Chemistry & Physics, University of Kwazulu-Natal, Durban 4001, South Africa., Cascone O; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina; Instituto Nacional de Producción de Biológicos, ANLIS Malbrán, Av. Vélez Sársfield 563, 1282 Buenos Aires, Argentina., Martínez Ceron MC; Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113 Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Junín 956, 1113 Buenos Aires, Argentina. Electronic address: camartinez@ffyb.uba.ar.
Jazyk: angličtina
Zdroj: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences [J Chromatogr B Analyt Technol Biomed Life Sci] 2020 Jun 01; Vol. 1146, pp. 122070. Date of Electronic Publication: 2020 Mar 16.
DOI: 10.1016/j.jchromb.2020.122070
Abstrakt: The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A 2 (PLA 2 ) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA 2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA 2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA 2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA 2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.
Competing Interests: Declaration of Competing Interest None.
(Copyright © 2020 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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