Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies.

Autor:	Almasia NI; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina. almasia.natalia@inta.gob.ar., Molinari MP; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina.; Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Godoy Cruz 2290, C1425FQB, Autonomous City of Buenos Aires, Argentina., Maroniche GA; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina.; Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata (UNMdP), km73,5 route 226, Balcarce, Buenos Aires, Argentina., Nahirñak V; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina., Barrios Barón MP; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina.; Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Godoy Cruz 2290, C1425FQB, Autonomous City of Buenos Aires, Argentina., Taboga OA; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina.; Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Godoy Cruz 2290, C1425FQB, Autonomous City of Buenos Aires, Argentina., Vazquez Rovere C; Instituto de Biotecnología, Centro de Investigación en Ciencias Veterinarias y Agronómicas, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional de Tecnología Agropecuaria, Repetto y De Los Reseros s/n, CP 1686, Hurlingham, Buenos Aires, Argentina.; Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Godoy Cruz 2290, C1425FQB, Autonomous City of Buenos Aires, Argentina.; LABINTEX-INTA, Agropolis Fondation, Montpellier, France.
Jazyk:	angličtina
Zdroj:	BMC biotechnology [BMC Biotechnol] 2017 Nov 09; Vol. 17 (1), pp. 75. Date of Electronic Publication: 2017 Nov 09.
DOI:	10.1186/s12896-017-0401-2
Abstrakt:	Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanum tuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal and antibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state using bacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility, misfolding or low yields. Results: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insect cells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinant peptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. An optimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gave rise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stable transgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinant antimicrobial peptides expression in other heterologous systems. Conclusions: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide in baculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploited for its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of the baculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functional analysis and antibody production, as an important step to both elucidate their accurate physiological role and to deepen the study of their biotechnological uses.
Databáze:	MEDLINE
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