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

© 2019 N. I. Hrunyk et al.; Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org/ and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 577.112.083

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 acti vity 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

INTRODUCTION
Plants synthesize various antimicrobial peptides (AMPs), that perform many biological functions. The AMPs as the main components of the plant's innate immune system are ubiquitous and found as host defense against pathogens and pests in diverse organisms ranging from microbes to animals [4,29]. Defensins form one of several families of plant AMPs [19,27,28].
Plant defensins are positively charged cysteine-rich peptides consisting of 45-54 amino acids. They contain a conservative CSαβ structural motif, consisting of one α-helix positioned on the top of three antiparallel β-sheets stabilized by 4-5 disulfide brid ges [11,[32][33][34]. Despite a conservative tertiary structure of these peptides, there is a substantial functional variation between them. 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 (fungi, bacteria, viruses), abiotic stress tolerance to salinity, drought, cold and metals (Zn, Cd) and in other, non-defense functions [1,16,20,21,23,26,30]. In addition, plant defensins can affect the fitness of insect pests by inhibiting their digestive enzymes such as proteases and amylases, as well as blocking ion channels [2]. Defensin of Cassia fistula seeds passesses trypsin inhibitory activity [31].
The multifunctional properties of defensin peptides make them attractive candidates for the design of transgenic plants resistant to phytopathogenic organisms or for the development of new drugs with antimicrobial properties [7,22,28]. However, to create an integrated platform for producing the antimicrobials or for the design of transgenic plants with specified properties, it is necessary to define the relationship between the structure and function of these molecules.
For detailed structural and functional studies of defensins, a significant amount of active peptides is needed. Purification of defensins from their original source, where their content is very low, is a time-consuming labor-intensive procedure, and the yield of target peptides is low. Therefore, large-scale production of purified active defensins is typically done in heterologous expression systems, usually using Escherichia coli [8]. Previously, we have employed this approach to obtain the recombinant defensin 1 from Scots pine for NMR structural analysis. PsDef1 is the first defensin of the coniferous plants whose NMR structure and properties have been thoroughly investigated [6,10,12,13,15]. It is worth mentioning that defensins in the pine genome are represen ted by a multigene family. Previously, we cloned several defensin genes including PsDef2 (Gen-Bank Acc. No. EF 455617.1) whose sequence has 90 % identity to PsDef1 (GenBank Acc. No. EF 455616.1) [14]. We hypothesize that the comparative characteristics of biological activities and features of three-dimensional structures of these two highly homologous pine defensins can reveal which structural motifs are involved in the performance of their functions. Isolation of an active PsDef2 peptide is an essential step in testing this hypothesis.
Here, we addressed the production, purification and subsequent determination of the in vitro antimicrobial activity of PsDef2.

Construction of expression vector and protein expression.
The construct for recombinant PsDef2 expression was designed by GenScript Biotech Corporation (USA). The construct containing PsDef2 cDNA encoding the mature form of the protein was inserted into pET-32 Ek/LIC vector (Novagen). The vector was used to transform E. coli BL21-CodonPlus(DE3)-RIL competent cells, as described in manufacturer's recommendations.
Isolated colonies were inoculated in LB medium (containing 100 µg/ml ampicillin and 50 µg/ml of chloramphenicol) and shaken overnight at 30 °C. Then, the overnight cell suspension was added to medium with appropriate antibiotics with a ratio of 2 % (v/v) at 37 ºC. To find optimal condition for TRX-PsDef2 protein expression different concentrations of IPTG, as well as time of protein expression were tested. When the culture reached an OD 600 of 0.8, protein expression was induced by the addition of IPTG at three concentrations: 0.1, 0.5 and 1.0 mM. Additionally, we tested the effect of temperature (37 ºC, 28 ºC and 25 ºC) on protein expression at four time points: 2, 3, 4 and 18 h. To monitor protein expression, non-induced cell culture was used. 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. Cells were disrupted in the lysis buffer containing 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 25 mM imidazole and 1 mM phenylmethanesulfonyl fluoride (PMSF) by sonication (QSonica Misonix XL-2000). Soni cation was performed on ice for 3 × 1 min at 50 % amplitude, pulsing at 1 s on /1 s off, and with breaks between cycles to prevent heating of the mixture and subsequent centrifugation at 12000 rpm for 20 min at 4 ºC. Total lysates and supernatants were analyzed by 12 % SDS-PAGE performed using Laemmli method [18].
Protein purification and analysis. TRX-PsDef2 fusion protein was purified using HisPurNi-NTA resin (Thermo Fisher, USA). Briefly, the resulting supernatant was added to a 50 % metal affinity sorbent, equilibrated with lysis buffer and incubated for 1 h at 4 °С. After incubation, the suspension was loaded in the column. Unbound proteins were removed by washing sorbent with 20 mM Tris-HCl buffer at pH 8.0 with 400 mM NaCl and 40 mM imidazole. Bound proteins were eluted from the column with 300 mM imidazole dissolved in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl. Then eluted fractions were analyzed by 12 % SDS-PAGE electrophoresis [18]. Fractions containing recombinant TRX-PsDef2 were pooled and dialyzed against buffer containing 20 mM Tris-HCl, pH 8.0, 50 mM NaCl. To remove 6His-TRX-tag, TRX-PsDef2 protein was cleaved using enterokinase (New England Biolabs, UK) at 18 ºC for 20 h in the recommended buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2 mM CaCl 2 ).
After cleavage, the digestion mixture was subjected to ion-exchange chromatography on a SP-Sepharose Fast Flow column (GE Healthcare) equilibrated with the 20 mM Tris-HCl, pH 8.0, 50 mM NaCl buffer. After extensive washing using the same buffer, bound proteins were eluted using step gradient from 0.05 to 1 M NaCl in the 20 mM Tris-HCl at pH 8.0. The elution of protein was monitored by absorbance at 280 nm and confirmed by 16.5 % SDS-PAGE. Fractions containing pure PsDef2, as determined by electrophoresis, with molecular mass about 6 kDa were collected, desalted, and concentrated by ultrafiltration with an Amicon Ultra 3K device (Millipore). Recovered Ps-Def2 peptide solution was tested against the phytopathogens.
Antimicrobial activity assay. Pathogenic F. sporotrichiella fungus and P. gonapodyides oomycete were inoculated on a PDA medium plate (90 × 15 mm Petri dish) and cultured at 25 ºC. When each colony reached 4 cm in diameter, 10 µL of 5 µM recombinant PsDef2 aqueous solution were added dropwise on the nutrient medium at 5 mm from the periphery of the microbial colony. As a blank control, 10 µL of sterile distilled water was applied. After the treatment, plates were cultured at 25 ºC, and the growth status of each phytopathogen was monitored within 2 days.

RESULTS AND DISCUSSION
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. Thioredoxin was selected as a fusion partner, because it helps in maintaining the expressed fusion protein in a soluble fraction and prevents its precipitation in inclusion bodies [5,17].
This construct was transformed into BL21-CodonPlus(DE3)-RIL E. coli cells for expression. Besides fast growth rate and low cost, this strain also offers IPTG-inducible protein expression. It was specially designed to resolve the codon bias problem to improve protein expression by supplying additional copies of specific tRNA genes that are rare in E. coli. The SDS-PAGE analysis of total lysates from induced and non-induced cells revealed the presence of recombinant protein with expected molecular weight of 22.8 kDa (Fig. 1, A). The fusion protein consists of 109 amino acids of thioredoxin protein at the N-terminus and 50 aa corresponding to mature form of PsDef2 at the C-terminus. There is also a 50 aa insert between TRX and PsDef2, that contains cleavable S-tag and His-tag sequences for protein detection and purification along with sites for thrombin and enterokinase proteases. In addition, this analysis has shown that TRX-PsDef2 fusion protein is soluble in buffer without any detergents. No peptide bands corresponding to the recombinant protein were observed in the non-induced cultures (Fig. 1, A). We also found that at chosen experimental setup, some TRX-PsDef2 protein was accumulated in inclusion bodies (data not shown). Therefore, we optimized the conditions for achieving maximal yield of soluble TRX-PsDef2. Expression of soluble fusion PsDef2 was optimized by varying IPTG concentrations (0.1-1 mM), temperatures (25 °C, 28 °C, 37 °C), and time of induction (Fig. 1, B). We found that optimal expression of soluble TRX-PsDef2 was achieved by the induction of bacterial culture with 0.5 mM IPTG for 3 h at 25 °C. Affinity purification of recombinant TRX-PsDef2 was carried out on HisPurNi-NTA column. As shown in Fig. 2, A (lanes 5-10), fractions eluted from the affinity column by step gradient of imidazole mainly contain a protein of approximately 23 kDa that closely correlates to the predicted molecular mass of the TRX-PsDef2 fusion protein.
Purified preparations of TRX-PsDef2 were pooled and dialyzed against the buffer containing 20 mM Tris-HCl, 50 mM NaCl, pH 8,0 (Fig. 2, B, line 1). To remove the TPXtag, fusion protein was subjected to proteolytic cleavage according to the manufacturer's recommendation. Initially, we performed a time-course digestion analysis with enterokinase. We found that the complete cleavage of the fusion protein TRX-PsDef2 is achieved after 20 h of incubation with the protease at room temperature (Fig. 2, B, line 2).
After enzymatic proteolysis, the reaction mixture contained defensin 2 and Ніstagged proteins. Repeated affinity purification on Ni-NTA resin is often used to remove proteins with His-tag from digestive mixture. According to our and other researchers' results (data not shown), this procedure is not effective for obtaining a desired product of high purity [3]. In our previous studies, Scots pine defensin1 was purified from seedlings, as well as from bacterial cells, using ion-exchange chromatography with a high yield of purity ≥ 95 % [12,13]. In order to isolate PsDef2 from the digested mixture, we used ion-exchange chromatography on a SP-Sepharose with a step gradient from 0.05 to 1 M NaCl. The electrophoretic profile of different fractions of the recombinant pine defensin 2 obtained during the purification process can be seen in Fig. 3. Thioredoxin detected in the flowthrough (lane 2). In addition, the enzyme/peptide mixture contained about 5% of the fusion protein TRX-PsDef2, which was eluted by 0.2-0.3 М NaCl, is shown in lanes 3-4.
The presence of PsDef2 was tested electrophoretically, and it was determined that it was contained in fractions that were eluted from the cation exchanger using a high ionic strength buffer from 0.4 to 0.8 M NaCl (lanes 5-9).
One of the proofs of the correct folding of the recombinant protein expressed in the prokaryotic system is the presence of expected biological activity. As shown earlier, defensin 1 from Scots pine, that is highly-homologous to PsDef2, exhibits high activity against a number of phytopathogenic fungi. We tested the biological activity of Scots pine defensin 2 against F. sporotrichiella and Ph. gonapodyides. The assay of antimicrobial activity revealed that PsDef2 manifested growth inhibitory activity against tested phytopathogens. Recombinant Scots pine defensin 2 at 5 μM the concentration inhibited hyphal growth and affected the shape of microbial colonies (Fig. 4).
Similar inhibitory activity of recombinant Scots pine defensin 1 against Heterobasidion annosum and Fusarium solani was shown in our previous studies. Therefore, there is a need for further studies of antibacterial and other activities of PsDef2. Since it belongs to the same group of Scots pine defensins as PsDef1 and is similar to PsDef1, it may demonstrate a broader spectrum of biological activity. The developed protocol resulted in the production of highly homogeneous recombinant defensin 2 that runs on the SDS-PAGE with a predicted molecular mass of 6 kDa (Fig. 3). By optimizing the expression conditions, affinity purification of recombinant TRX-PsDef2 and removing of TRX moiety from the fusion protein by proteolytic cleavage, as well as ion-exchange chromatography on strong cation exchanger, we managed to produce 1mg of PsDef2 per liter of bacterial culture. Importantly, the developed protocol results in the production of highly stable and biologically active PsDef2.
The use of a prokaryotic system for the production of bioactive plant defensins is challenged by a correct pairing of four disulfide bridges, which is a characteristic feature of these peptides. To enhance disulfide bond formation in bacteria cytoplasm, active plant defensins were produced as translational fusion proteins with thioredoxin in Origami E. coli strain [9,25]. Pervieux et al. (2004) used this system for the production of a bioactive defensin from Picea glauca that exhibits 80 % identity to PsDef2 [24]. We found that recombinant PsDef2 possesses antimicrobial activity at 5 µM concentration. These results suggest that pET32/BL21 CodonPlus(DE3)-RIL expression system is also efficient for the production of bioactive plant defensins. The availability of recombinant PsDef2 provides a possibility not only to examine its antimicrobial properties but also to study its structure by spectroscopic methods (circular dichroism, NMR) to establish the relationship between the structure and function of pine defensins.
In conclusion, this work describes a successful heterologous expression of plant defensin with antimicrobial activity against phytopathogens. The antimicrobial activity of the recombinant PsDef2 warrants further investigation of its potential in biotechnology applications for protecting plants against infectious diseases.