Biochemical analyses of ferns have predominantly focused on investigating their secondary metabolites rather than proteins. Of particular interest are insecticidal proteins found in ferns, which hold potential for use as bioinsecticides. In this study, we aimed to identify the presence of pore-forming proteins in fern species growing in Slovenia. To achieve this, we prepared their aqueous extracts and exposed them to mammalian (bovine erythrocytes) and insect (Sf9 cell line) cellular models to assess whether the selected species induce cell lysis. Furthermore, we investigated whether the proteins from ferns bind to the membrane lipids. This would indicate that the lytic activities observed in the aqueous extracts are direct results of the activities of membrane-active proteins. Through screening analysis, we confirmed the presence of cytolytic proteins in ferns and determined that 89 % of the tested species exhibited a lytic effect on insect cells. In eight of these species, lytic activity was specifically attributed to protein activity, suggesting their potential as bioinsecticidal agents with biotechnological relevance. Among them, Lycopodium annotinum and Equisetum variegatum demonstrated the highest potential in terms of lytic and cytotoxic activity, likely due to the presence of membrane-active proteins. To determine the amino acid sequences of these proteins, we employed mass spectrometry. However, the analysis did not yield the expected results, which we attribute to the limited availability of fern genome databases.
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