PROKARYOTIC EXPRESSION AND PURIFICATION OF BIOACTIVE DEFENSIN 2 FROM PINUS SYLVESTRIS L.
DOI: http://dx.doi.org/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 (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.
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1. Ali S., Ganai B.A., Kamili A.N., Bhat A.A., Mir Z.A., Bhat J.A., Tyagi A., Islam S.T., Mushtaq M., Yadav P., Rawat S., Grover A. Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance. Microbiol. Res., 2018; 212-213: 29-37. Crossref ● PubMed ● Google Scholar | ||||
| ||||
2. Bloch C., Jr., Richardson M. A new family of small (5 kDa) protein inhibitors of insect α-amylases from seeds or sorghum (Sorghum bicolor (L.) Moench.) have sequence homologies with wheat γ-purothionins. FEBS Lett., 1991; 279: 101-104. Crossref ● PubMed ● Google Scholar | ||||
| ||||
3. Dos Santos I.S., Carvalho Ade O., de Souza-Filho G.A., do Nascimento V.V., Machado O.L., Gomes V.M. Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect α-amylase inhibitory activity. Protein Expr. Purif., 2010; 71: 8-15. Crossref ● PubMed ● Google Scholar | ||||
| ||||
4. Egorov T.A., Odintsova T.I., Pukhalsky V.A., Grishin E.V. Diversity of wheat anti-microbial peptides. Peptides, 2005; 26: 2064-2073. Crossref ● PubMed ● Google Scholar | ||||
| ||||
5. Elmorjani K., Lurquin V., Lelion A, Rogniaux H., Marion D. A bacterial expression system revisited for the recombinant production of cystein-rich plant lipid transfer proteins. Biochem Biophys Res Commun, 2004; 316: 1202-1209. Crossref ● PubMed ● Google Scholar | ||||
| ||||
6. Ermakova E.A., Faizullin D.A., Idiyatullin B.Z. Khairutdinov B.I., Mukhamedova L.N., Tarasova N.B., Toporkova Y.Y., Osipova E.V., Kovaleva V., Gogolev Y.V., Zuev Y.F., Nesmelova I.V. Structure of Scots pine defensin 1 by spectroscopic methods and computational modeling. Int. J. Biol. Macromol., 2016; 84: 142-152. Crossref ● PubMed ● Google Scholar | ||||
| ||||
7. Gao A.G., Hakimi S.M., Mittanck C.A., Wu Y., Woerner B.M., DStark M., Shah D.M., Liang J.H., Rommens C.M.T. Fungal pathogen protection in potato by expression of a plant defensin peptide. Nature Biotechnology, 2000; 18: 1307-1310. Crossref ● PubMed ● Google Scholar | ||||
| ||||
8. Gazzaneo L.R.S., Pandolfi V., de Jesus A.L.S., Crovella S., Benko-Iseppon A. M., de Freitas A.C. Heterologous expression systems for plant defensin expression: examples of success and pitfalls. Curr Protein Pept Sci., 2017; 18 (4): 391-399. Crossref ● PubMed ● Google Scholar | ||||
| ||||
9. Kaomek M., Mizuno K., Fujinira T., Sriyotha P., Cairna J.R.K. Cloning, expression and characterization of an antifungal chitinase from Leucaena leucocephla de Wit. Biosci. Biotechnol. Biochem., 2003; 67: 667-676. Crossref ● PubMed ● Google Scholar | ||||
| ||||
10. Khairutdinov B.I., Ermakova E., Yusypovych Y.M., Bessolicina E.K., Tarasova N.B., Toporkova Y.Y., Kovaleva V., Zuev Y.F., Nesmelova I.V. NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris. Biochimica et biophysica acta. Proteins and proteomics, 2017; 1865(8): 1085-1094. Crossref ● PubMed ● Google Scholar | ||||
| ||||
11. Kobayashi Y., Sato A., Takashima H., Tamaoki H., Nishimura S., Kyogoku Y., Ikenaka K., Kondo T., Mikoshiba K., Hojo H., Aimoto S., Moroder L. A new α-helical motif in membrane active peptides. Neurochem. Internat, 1991; 18: 525-534. Crossref ● Google Scholar | ||||
| ||||
12. Kovaleva V.A., Kiyamova R.G., Cramer R., Krynytskyy H. T., Gout I.T., Filonenko V.V., Gout R. Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings. Рeptides, 2009; 30(12): 2136-2143. Crossref ● PubMed ● Google Scholar | ||||
| ||||
13. Kovaleva V.A., Krynytskyy H.T., Gout I.I., Gout R.T. Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1. Appl. Microbiol. Biotechnol., 2011; 89(4): 1093-1101. Crossref ● PubMed ● Google Scholar | ||||
| ||||
14. Kovalyova V.A., Gout I.T. Molecular cloning and characterization of Scotch pine defensin 2. Cytology and Genetics, 2008; 42(6): 408-412. Crossref ● Google Scholar | ||||
| ||||
15. Kovalyova V.A., Gout I.T., Kiyamova R.G., Filonenko V.V., Gout R.T. Cloning and analysis of defensin 1 cDNA from Scots pine. Biopolymers and Cell, 2007; 23(5): 398-404. Crossref ● Google Scholar | ||||
| ||||
16. Lacerda A.F., Vasconcelos É.A.R., Pelegrini P.B., Grossi de Sa M.F. Antifungal defensins and their role in plant defense. Frontiers in Microbiology, 2014; 5: 116. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
17. La Vallie E.R., Di Blasio-Smith E.A., Collins-Racie L.A., Lu Z., McCoy J.M. Thioredoxin and related proteins as multifunctional fusion tags for soluble expression in E. coli. Methods MolBiol, 2003; 205: 119-140. Crossref ● PubMed ● Google Scholar | ||||
| ||||
18. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227: 680-685. Crossref ● PubMed ● Google Scholar | ||||
| ||||
19. Lay F.T, Anderson M.A. Defensins - components of the innate immune system in plants. Curr. Protein Pept Sci., 2005; 6: 85-101. Crossref ● PubMed ● Google Scholar | ||||
| ||||
20. Luo J.S., Gu T.Y., Yang Y., Zhang Z.H. A non-secreted plant defensin AtPDF26 conferred cadmium tolerance via its chelation in Arabidopsis. Plant Molecular Biology, 2019; 100(4-5): 561-569. Crossref ● PubMed ● Google Scholar | ||||
| ||||
21. Mirouze M., Sels J., Richard O., Czernic P., Loubet S., Jacquier A., Francois I.E., Cammue B.P., Lebrun M., Berthomieu P., Marques L. A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance. Plant J., 2006; 47: 329-42. Crossref ● PubMed ● Google Scholar | ||||
| ||||
22. Nordström R., Malmsten M. Delivery systems for antimicrobial peptides. Adv. Colloid Interface Sci. 2017; 242: 17-34. Crossref ● PubMed ● Google Scholar | ||||
| ||||
23. Parisi K., Shafee T.M.A., Quimbar P., van der Weerden N.L., Bleackley M.R., Anderson M.A. The evolution, function and mechanisms of action for plant defensins. Seminars in Cell & Developmental Biology, 2019; 88: 107-118. Crossref ● PubMed ● Google Scholar | ||||
| ||||
24. Pervieux I., Bourassa M., Laurans F., Hamelin R.C., Seguin A. A spruce defensin showing strong antifungal activity and increased transcript accumulation after wounding and jasmonate treatments. Physiological and Molecular Plant Pathology, 2004; 64: 331-341. Crossref ● Google Scholar | ||||
| ||||
25. Picart P., Pirttilä A.M., Raventos D. Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity. BMC Plant Biol., 2012; 12: 180. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
26. Sher Khan R., Iqbal A., Malak R., Shehryar K., Attia S., Ahmed T., Khan M.A., Arif M., Mii M. Plant defensins: types, mechanism of action and prospects of genetic engineering for enhanced disease resistance in plants. 3 Biotech., 2019; 9(5): 192-204. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
27. Sinha M., Singh R.P., Kushwaha G.S., Iqbal N., Singh A., Kaushik S., Kaur P., Sharma S., Singh T.P. Current overview of allergens of plant pathogenesis related protein families. Sci. World J., 2014; 2014: 543195. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
28. Sinha R, Shukla P. Antimicrobial Peptides: Recent insights on biotechnological interventions and future perspectives. Protein Pept Lett., 2019; 26 (2): 79-87. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
29. Sitaram N. Antimicrobial peptides with unusual amino acid compositions and unusual structures. Curr. Med. Chem., 2006; 13(6): 679-696. Crossref ● PubMed ● Google Scholar | ||||
| ||||
30. Skolotneva E. S., Salina E. A. Resistance mechanisms involved in complex immunity of wheat against rust diseases. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov Journal of Genetics and Breeding, 2019; 23(5): 542-550. (In Russian) Crossref ● Google Scholar | ||||
| ||||
31. Wijaya R., Neumann G.M., Condron R., Hughes A.B., Polya G.M. Defense proteins from seed of Сassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin. Plant Sci., 2000; 159: 243-255. Crossref ● Google Scholar | ||||
| ||||
32. Yount N. Y., and Yeaman M. R. Multidimensional signatures in antimicrobial peptides. Proc. Natl. Acad. Sci. U.S.A., 2004; 101: 7363-7368. Crossref ● PubMed ● PMC ● Google Scholar | ||||
| ||||
33. Yount N.Y., Andrés M.T., Fierro J.F., Yeaman M.R. The γ-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins. Biochim. Biophys. Acta, 2007; 1768: 2862-2872. Crossref ● PubMed ● Google Scholar | ||||
| ||||
34. Zhu S., Gao B., Tytgat J. Phylogenetic distribution, functional epitopes and evolution of the CSαβ superfamily. Cell Mol. Life Sci., 2005; 62: 2257-2269. Crossref ● PubMed ● Google Scholar |
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