Prokaryotic expression and purification of bioactive defension 2 from Pinus sylvestris L.

I V Nesmelova,Yu M Yusypovych,N I Hrunyk,Yu I Shalovylo,I I Roman,V A Kovaleva

doi:10.30970/sbi.1302.603

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 ( Ps Def1) demonstrating high activity against fungi and bacteria. Importantly, Ps Def1 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. ( Ps Def2), whose sequence has 90 % identity to Ps Def1. We used pET32/BL21-CodonPlus (DE3)-RIL Escherichia coli expression system to produce large quantities of the recombinant Ps Def2 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- Ps Def2 on HisPurNi-NTA resin, the fusion protein was subjected to proteolytic cleavage by enterokinase. Ps Def2 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 Ps Def2 was higher than 95 %, as verified by 16.5 % SDS-PAGE. The recombinant peptide Ps Def2 showed activity against phytopathogenic Fusarium sporotrichiela fungus and Phythophtora gonapodyides oomycete at 5 µM concentration. The availability of recombinant Ps Def2 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. Keywords : defensins, Scots pine, heterologous expression, protein purification, antimicrobial activity

Highlights

Plants synthesize various antimicrobial peptides (AMPs), that perform many biological functions
Based on in vitro assays of isolated peptides, gene expression analysis and studies of transgenic plants, defensins were suggested to be involved in defense against the phytopathogenic organisms, abiotic stress tolerance to salinity, drought, cold and metals (Zn, Cd) and in other, non-defense functions [1, 16, 20, 21, 23, 26, 30]
To follow the heterologous expression of Scots pine defensin 2 gene, the cDNA sequence encoding PsDef2 fragment was inserted in the pET-32 Ek/LIC (Novagen), linearized in a frame as a fusion with the thioredoxin

Summary

Introduction

Plants synthesize various antimicrobial peptides (AMPs), that perform many biological functions. After finding the optimal conditions, TRX-PsDef2 fusion protein expression was induced with 0.5 mM of IPTG at 25 oC, and cells were harvested after 3 h post-induction by centrifugation at 10000 × g for 10 min at 4 oC.

Results

Conclusion