Expression 0and purification of the recombinant cysteine-rich antimicrobial peptide Snakin-1 in baculovirus infected insect cells.

ALMASIA, NATALIA INÉS; MOLINARI MP; ; MARONICHE G.; ; NAHIRÑAK V; ; BARRIOS BARON MP.; ; TABOGA O;; VAZQUEZ ROVERE C

BMC BIOTECHNOLOGY

BIOMED CENTRAL LTD

Lugar: Londres; Año: 2017

1472-6750

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 detected in the nuclear fraction of infected Sf9 cells. An optimized purification procedure allowed the production rSN1 that was used for immunization of mice, giving rise to polyclonal antibodies that were able to 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. Given the important function of Snakin/GASA proteins, the use of the baculovirus expression system for obtaining high-level protein expression, for biologicalassays, structural and functional analysis and antibody production, is an important step towards elucidating their accurate physiological role and to deepen the study of its biotechnological uses.