In silico Analysis and Expression of Osmotin-EAAAK-LTP Fused Protein

Document Type : Research Article


National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, IRAN


Antifungal agents are causing different problems in the agriculture industry. Plants are using various defense mechanisms for resistance against fungal pathogens. Some examples of these mechanisms are making physical barriers, producing chemical components and pathogenesis-related proteins such as lipid transfer protein (LTP) and Osmotin which can inhibit the growth of fungi at micro-molar concentrations. In this study, Osmotin and LTp genes were fused by the EAAAK linker to produce a single-fused gene construct. An in silico approach was used to predict and analyze Osmotin-EAAAK-LTP fused protein. Secondary and tertiary structure and mRNA formation of fused protein were predicted using bioinformatics tools. The designed construct was chemically synthesized and cloned in the pUC57 cloning vector. To express the fused protein gene was subcloned in expression vector pET-21b (+) with a hexahistidine tag. This gene was used for prokaryotic expression in E. coliBL21 (DE3) host. Different expression conditions were examined for expressing of fused protein. The fused protein was expressed with 1 mM IPTG after 3 hours of incubation at 28°C. The expression of 36.5 kDa protein was confirmed by western blotting. The study of antifungal activity of expressed fused protein was achieved by radial diffusion assay. This protein was able to exhibit antifungal activity towards experimented plant pathogenic fungi under in vitro conditions.


Abad LR, D'Urzo MP, Liu D, Narasimhan ML, Reuveni M, Zhu JK, Bressan RA. 1996. Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization. Plant Sci 118(1): 11-23.
Amet N, Lee HF, Shen W. 2009. Insertion of the designed helical linker led to increased expression of tf-based fusion proteins. Pharm Res 26(3): 523-528.
Arai R, Wriggers W, Nishikawa Y, Nagamune T, Fujisawa T. 2004. Conformations of variably linked chimeric proteins evaluated by synchrotron X‐ray small‐angle scattering. Proteins 57: 829-838.
Aroul-Selvam R, Hubbard T, Sasidharan R. 2004. Domain insertions in protein structures. J Mol Biol 338(4): 633-641.
Bai Y, Shen WC. (2006). Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm Res 23(9): 2116-2121.
Blilou I, Ocampo JA, García‐Garrido, JM. 2000. Induction of Ltp (lipid transfer protein) and Pal (phenylalanine ammonia‐lyase) gene expression in rice roots colonized by the arbuscular mycorrhizal fungus Glomus mosseae. J Exp Bot 51(353): 1969-1977.
Brogue K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Broglie R. 1991. Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254(5035): 1194-1197.
Chen X, Bai Y, Zaro JL, Shen WC. 2010. Design of an in vivo cleavable disulfide linker in recombinant fusion proteins. Biotechniques 49(1): 513-518.
Carvalho Ade O, Gomes VM. 2007. Role of plant lipid transfer proteins in plant cell physiology-a concise review. Peptides 28(5): 1144-1153.
Douliez JP, Pato C, Rabesona H, Mollé D,  Marion D. 2001. Disulfide bond assignment, lipid transfer activity and secondary structure of a 7‐kDa plant lipid transfer protein, LTP2. Eur J Biochem 268(5): 1400-1403.
Edreva A. 2005. Pathogenesis-related proteins: research progress in the last 15 years. Gen Appl Plant Physiol 31(1-2): 105-124.
Freeman BC, Beattie GA. 2008. An overview of plant defenses against pathogens and herbivores. The Plant Health Instr 6(11). doi: 10.4172/2157-7471.1000322.
Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A. 2005. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook, Springer.
Kader JC. 1996. Lipid-transfer proteins in plants. Annu Rev Plant Biol 47(1): 627-654.
Karri V, Bharadwaja KP. 2013. Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein. Funct Integr Genomics 13(4): 435-443.
Kim H, Mun JH, Byun BH, Hwang HJ, Kwon YM, Kim SG. 2002. Molecular cloning and characterization of the gene encoding osmotin protein in Petunia hybrida. Plant Sci 162(5): 745-752.
Lee M, Bang K, Kwon H, Cho S. 2013. Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker. Mol Biol Rep 40(6): 3953-3960.
Li HP, Zhang JB, Shi RP, Huang T, Fischer R, Liao YC. 2008. Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusarium-specific antibody and an antifungal peptide. Mol Plant Microbe interact 21(9): 1242-1248.
Liu D, Rhodes D, D'Urzo MP, Xu Y, Narasimhan ML, Hasegawa PM, Abad L. 1996. In vivo and in vitro activity of truncated osmotin that is secreted into the extracellular matrix. Plant Sci 121(2): 123-131.
Lorito M, Woo SL, D'ambrosio M, Harman GE, Hayes CK, Kubicek CP, Scala F. 1996. Synergistic interaction between cell wall degrading enzymes and membrane affecting compounds. Mol Plant Microbe Interact  9(3): 206-213.
Makrides SC. 1996. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Mol Biol Rev 60(3) 512-538.
Mani T, Sivakumar KC, Manjula S. 2012. Expression and functional analysis of two osmotin (PR5) isoforms with differential antifungal activity from Piper colubrinum: prediction of structure-function relationship by bioinformatics approach. Mol biotechnol 52(3): 251-261.
Min K, Ha SC, Hasegawa PM, Bressan RA, Yun DJ, Kim KK. 2004. Crystal structure of osmotin, a plant antifungal protein. Proteins 54(1): 170-173.
Narasimhan ML, Coca MA, Jin J, Yamauchi T, Ito Y, Kadowaki T, Hasegawa PM. 2005. Osmotin is a homolog of mammalian adiponectin and controls apoptosis in yeast through a homolog of mammalian adiponectin receptor. Mol cell 17(2): 171-180.
Osusky M, Osuska L, Hancock RE, Kay WW, Misra S. 2004. Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot. Transgenic Res 13(2): 181-190.
Patkar RN, Chattoo BB. 2006. Transgenic indica rice expressing ns-LTP-like protein shows enhanced resistance to both fungal and bacterial pathogens. Mol Breeding 17(2): 159-171.
Regente MC, De La Canal L. 2000. Purification, characterization and antifungal properties of a lipid‐transfer protein from sunflower (Helianthus annuus) seeds. Physiol Plant 110(2): 158-163.
Sarowar S, Kim YJ, Kim KD, Hwang BK, Ok SH, Shin JS. 2009. Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco. Plant Cell Rep 28(3): 419-427.
Singh NK, Bracker CA, Hasegawa PM, Handa AK, Buckel S, Hermodson MA, Bressan RA. 1987. Characterization of osmotin: a thaumatin-like protein associated with osmotic adaptation in plant cells. Plant Physiol 85(2): 529-536.
Tulasi RB, Nadimpalli SK. 1997. Purification of α‐mannosidase activity from Indian lablab beans. Biochem Mol Biol Int 41(5): 925-931.
Uhlén M, Forsberg G, Moks T, Hartmanis M, Nilsson B. 1992. Fusion proteins in biotechnology. Curr Opin Biotechnol 3(4): 363-369.
Van Loon LC, Van Strien EA. 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant P 55(2): 85-97.
Wriggers W, Chakravarty S, Jennings PA. 2005. Control of protein functional dynamics by peptide linkers. Biopolymers 80(6): 736-746.
Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, Santora L. 2007. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 25(11): 1290-1297.
Yang J, Zhang Y. 2015. I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Research 43(W1): W174-W181.
Yu K, Liu C, Kim BG, Lee DY. 2015. Synthetic fusion protein design and applications. Biotechnol Adv 33(1): 155-164.
Zhang J, Yun J, Shang Z, Zhang X, Pan B. 2009. Design and optimization of a linker for fusion protein construction. Prog Nat Sci 19(10): 1197-1200.