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 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.

Keywords: Antimicrobial peptide; Baculovirus expression system; Cysteine-rich; GASA; Snakin-1; Spodoptera frugiperda Sf9 cells.

Fig. 1

Subcellular localization of rSN1 in nuclei of Sf9 cells. StSN1 fused to GFP by the N-terminal end was transiently expressed in Sf9 cells and examined by live fluorescent imaging. Nucleus was visualized by mCherry-based fluorescent marker developed by Maroniche et al. [35]. In each case, images of the bright field (BF), signal of the fluorescent fusion with (SN1SP-GFP) or without the signal peptide (SN1ΔSP-GFP), nuclear marker and the merge of the images are shown. Scale bars: 10 μm

Fig. 2

Production of recombinant protein in SF9 insect cells over time. Western blot analysis using an anti-HIS antibody (SDS-PAGE 10%) of crude extracts at different days after infection. (−) = mock-infected Sf9 cells extract. M: PageRuler Prestained Protein Ladder (Thermo Scientific). DPI: Days post infection

Fig. 3

Production of recombinant Snakin-1 protein. Western Blot assay employing two independent recombinant virus (7A₂ and 7B₃ from the second and the third passage, respectively) and anti-His antibody. Concentrated supernatant, concentrated cytoplasm and nuclear fraction were evaluated. M: BenchMark Pre-Stained Protein Ladder. SDS-PAGE: 13.5%

Fig. 4

Purification steps of the rSN1 fusion protein. Upper panel: Western blot analysis using an anti-HIS antibody. Lane 1: Total nuclear lysate, Lane 2: Unbound proteins flowed through the column. Lanes 3–4: Proteins eluted from wash buffers of different stringent conditions tested (20 mM or 30 mM imidazole respectively). Lanes 5–6: Proteins eluted from acidic buffers of different pH 5.9 (lane 5) or pH 4.5 (lane 6). Lane 7: Proteins eluted (up to 6.3 mg/ml) with high concentrations of imidazole (500 mM). Lower panel: The nitrocellulose membrane stained with Ponceau dye for protein detection after the 10% SDS-PAGE analysis of representative purification steps of the rSN1. M: BenchMark Pre-Stained Protein Ladder. The arrow indicates the rSN1 peptide

Fig. 5

Scheme of inoculation and serum obtention. a Mice were immunized at Day 1, a booster dose was given at Day 23, a serum sample was taken at Day 30, an additional booster dose of incomplete Freund’s adjuvant (IFA) emulsion was given at Day 40 by employing extract nuclei expressing rSN1; and total serum was collected at Day 53. b Western blot analysis of extract nuclei of Sf9 cells either expressing rSN1 (+) or not (−). M: PageRuler Prestained Protein Ladder. Three different antibodies were used to reveal commercial anti-HIS, serum sample and total polyclonal serum (15% SDS-PAGE). The arrow indicates the rSN1 peptide

Fig. 6

Detection of SN1::GFP expressed by agroinfiltration in N. benthamiana plants. SN1ΔSP-Egfp: Protein extract from agroinfiltrated N. benthamiana with a vector capable of expressing SN1::GFP (~34 kDa); Control: Protein extract of N. benthamiana agroinfiltrated with an empty vector; M: BenchMark Pre-Stained Protein Ladder. Western blot analysis revealed either with Anti GFP or the anti-rSN1 polyclonal serum is shown, 10% SDS-PAGE. The arrow indicates the SN1::GFP fusion protein