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

Abstract

Background: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanumtuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal andantibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state usingbacterial, 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) insectcells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinantpeptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. Anoptimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gaverise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stabletransgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinantantimicrobial 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 inbaculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploitedfor its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of thebaculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functionalanalysis and antibody production, as an important step to both elucidate their accurate physiological role and todeepen the study of their biotechnological uses.