PROKARYOTIC EXPRESSION AND PURIFICATION OF BIOACTIVE DEFENSIN 2 FROM PINUS SYLVESTRIS L.

Abstract

Plant defensins are highly stable cysteine-rich peptides consisting of 45–54 amino acid residues with a characteristic conservative βαββ structure stabilized by 4–5 disulfide bridges. These peptides are key molecules of innate immune system in plants. They inhibit growth of many phytopathogenic fungi, and some of them exhibit antibacterial acti­vity. Defensins also possess other biological functions. The multifunctional properties of the defensin peptides make them attractive candidates for creation of new remedies with antimicrobial properties. Elucidation of nature of the structural and functional relationships in the antimicrobial peptides is an essential step in the development of drugs with acti­vity against pathogens. Previously, we have purified endogenous and recombinant Scots pine defensin 1 (PsDef1) demonstrating high activity against fungi and bacteria. Importantly, PsDef1 is the first defensin from coniferous plants whose NMR structure and pro­perties have been thoroughly investigated, also by the authors of this work. In this study, we presented the expression and affinity purification of recombinant defensin 2 from Pinus sylvestris L. (PsDef2), whose sequence has 90 % identity to PsDef1. We used pET32/BL21-CodonPlus (DE3)-RIL Escherichia coli expression system to produce large quantities of the recombinant PsDef2 peptide conjugated to thioredoxin (TRX). We found that the highest yield of recombinant protein in its soluble form was obtained at 0.5 mM of isopropyl-β-D-thiogalactoside (IPTG) concentration for 3 h of induction at 25 °С. After isolation of TRX-PsDef2 on HisPurNi-NTA resin, the fusion protein was subjected to proteolytic cleavage by enterokinase. PsDef2 was separated from the proteolytic fragments using the ion exchange on the SP-Sepharose Fast Flow column and a step gradient of 0.05–1 M NaCl. The purity of obtained recombinant PsDef2 was higher than 95 %, as verified by 16.5 % SDS-PAGE. The recombinant peptide PsDef2 showed activity against phytopathogenic Fusarium sporotrichiela fungus and Phythophtora gonapodyides oomycete at 5 µM concentration. The availability of recombinant PsDef2 gives an option not only to examine its antimicrobial properties but to study its structure by spectroscopic methods (circular dichroism, NMR) in order to esta­blish relationships between the structure and function of pine defensins.