STRUKTURNO MODELIRANJE, IZRAŽAVANJE I PREČIŠĆAVANJE HIMERIČNE KITINAZE42 KOJA SADRŽI HIS-TAG U SISTEMU DLAKASTOG KORENOVA NIKOTIANA TABACUM
STRUKTURNO MODELIRANJE, IZRAŽAVANJE I PROČIŠĆAVANJE
Sažetak
Himerna hitinaza42 (Chit42 koja sadrži ChBD) ima veliki potencijal kao kandidat za varenje i reciklažu hitina kao korisnog hranljivog materija, koji se može proizvesti u bioreaktorima. Biljka je jedan od efikasnih bioreaktora koji može da proizvodi eukariotske proteine u aktivnim oblicima. Odgovarajuću platformu za jednostavnu, ekonomičnu i brzu ekspresiju različitih rekombinantnih proteina obezbeđuje sistem dlakavog korena biljke. Zbog ogromne količine proteina u biljkama, His-tag se može koristiti za olakšavanje prečišćavanja rekombinantnih proteina. U ovom istraživanju korišćeni su različiti kompjuterski programi za analizu trodimenzionalne strukture himerne hitinaze42 koja sadrži His-tag. Rezultati su pokazali da ova uporedna modeliranja imaju izuzetan stepen tačnosti u predviđanju strukture fuzionisanog proteina. Z-skor od -9,38 i -3,64 predviđen za chit42 i CHBD od strane ProSA predstavlja dobar kvalitet modela. Takođe, bioinformatička zapažanja su pokazala da je His-tag bio izložen i da se može koristiti za prečišćavanje himerne hitinaze42. Himerna hitinaza42 koja sadrži His-tag je eksprimirana u Nicotiana tabacum dlakavim korenima, a istražena je uloga His-tag-a u detekciji vestern blot-om i prečišćavanjem pomoću Ni-NTA kolone. Prisustvo himerne hitinaze42 je verifikovano SDS-PAGE i Vestern blot analizom ekstrakta korena. Korak prečišćavanja je postignut korišćenjem His-tag i Ni-NTA kolone. Biološka aktivnost himerne hitinaze 42 biljnog porekla je potvrđena ispitivanjem hitinazne aktivnosti prečišćenog proteina na medijskoj ploči koja sadrži koloidni hitin.
Reference
Akeed, Y., Atrash, F., & Naffaa, W. (2020). Partial purification and characterization of chitinase produced by Bacillus licheniformis B307. Heliyon, 6(5), e03858.
Amarasinghe, C., & Jin, J.-P. (2015). The use of affinity tags to overcome obstacles in recombinant protein expression and purification. Protein and peptide letters, 22(10), 885-892.
Arakane, Y., Zhu, Q., Matsumiya, M., Muthukrishnan, S., & Kramer, K. J. (2003). Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect biochemistry and molecular biology, 33(6), 631-648.
Aslantas, Y., & Surmeli, N. B. (2019). Effects of N-terminal and C-terminal polyhistidine tag on the stability and function of the thermophilic P450 CYP119. Bioinorganic chemistry and applications, 2019.
Ataei, A., Zamani, M., Motallebi, M., Haghbeen, K., Ziaei, M., & Jourabchi, E. (2016). Increased antifungal activity of Chit42 from Trichoderma atroviride by addition of a chitin binding domain. Tropical Plant Pathology, 41(6), 350-356.
Barboza-Corona, J., Contreras, J., Velázquez-Robledo, R., Bautista-Justo, M., Gómez-Ramírez, M., Cruz-Camarillo, R., & Ibarra, J. (1999). Selection of chitinolytic strains of Bacillus thuringiensis. Biotechnology Letters, 21(12), 1125-1129.
Bhattacharya, D., Nagpure, A., & Gupta, R. K. (2007). Bacterial chitinases: properties and potential. Critical reviews in biotechnology, 27(1), 21-28.
Binod, P., Akanksha, K., Pandey, A., & Karthik, N. (2014). Production, purification and properties of fungal chitinases-A review.
Blaak, H., & Schrempf, H. (1995). Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. European journal of biochemistry, 229(1), 132-139.
Borisjuk, N. V., Borisjuk, L. G., Logendra, S., Petersen, F., Gleba, Y., & Raskin, I. (1999). Production of recombinant proteins in plant root exudates. Nature biotechnology, 17(5), 466-469.
Cardon, F., Pallisse, R., Bardor, M., Caron, A., Vanier, J., Ele Ekouna, J. P., . . . Guillet, M. (2019). Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha‐L‐iduronidase. Plant biotechnology journal, 17(2), 505-516.
Churklam, W., & Aunpad, R. (2020). Enzymatic characterization and structure-function relationship of two chitinases, LmChiA and LmChiB, from Listeria monocytogenes. Heliyon, 6(7), e04252.
Deng, J.-J., Shi, D., Mao, H.-h., Li, Z.-w., Liang, S., Ke, Y., & Luo, X.-c. (2019). Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion. International journal of biological macromolecules, 134, 113-121.
Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Duo-Chuan, L. (2006). Review of fungal chitinases. Mycopathologia, 161(6), 345-360.
Fan, Y., Fang, W., Guo, S., Pei, X., Zhang, Y., Xiao, Y., . . . Pei, Y. (2007). Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase. Applied and Environmental Microbiology, 73(1), 295-302.
Ghiasi-sis, J. G. ( 2016). The evaluation of transient expression of chimericchit42 containing ChBD in C-terminal under the control of SP-DDEE synthetic inducible promoter. Master thesis, National Institute of Genetic Engineering and Biotechnology.
Guthrie, J. L., Khalif, S., & Castle, A. J. (2005). An improved method for detection and quantification of chitinase activities. Canadian journal of microbiology, 51(6), 491-495.
Gutierrez-Valdes, N., Häkkinen, S. T., Lemasson, C., Guillet, M., Oksman-Caldentey, K.-M., Ritala, A., & Cardon, F. (2020). Hairy root cultures—a versatile tool with multiple applications. Frontiers in plant science, 11, 33.
Hang, Q., Woods, L., Feiss, M., & Catalano, C. E. (1999). Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins. Journal of Biological Chemistry, 274(22), 15305-15314.
Hayes, C. K., Klemsdal, S., Lorito, M., Di Pietro, A., Peterbauer, C., Nakas, J. P., . . . Harman, G. E. (1994). Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum. Gene, 138(1-2), 143-148.
Hung, M., Gibbs, C. S., & Tsiang, M. (2002). Biochemical characterization of rhinovirus RNA-dependent RNA polymerase. Antiviral research, 56(2), 99-114.
Kenig, M., Peternel, Š., Gaberc-Porekar, V., & Menart, V. (2006). Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins. Journal of Chromatography A, 1101(1-2), 293-306.
Kowolik, C. M., & Hengstenberg, W. (1998). The lactose transporter of Staphylococcus aureus: Overexpression, purification and characterization of the histidine‐tagged domains IIC and IIB. European journal of biochemistry, 257(2), 389-394.
Kowsari, M., Motallebi, M., & Zamani, M. (2014). Protein engineering of chit42 towards improvement of chitinase and antifungal activities. Current microbiology, 68(4), 495-502.
Kowsari, M., Zamani, M., & Motallebi, M. (2016). Overexpression of chimeric chitinase42 enhanced antifungal activity of Trichoderma harzianum against Fusarium graminearum. Mycologia Iranica, 3(1), 15-23.
Lico, C., Chen, Q., & Santi, L. (2008). Viral vectors for production of recombinant proteins in plants. Journal of cellular physiology, 216(2), 366-377.
Limón, M. C., Margolles-Clark, E., Benítez, T., & Penttilä, M. (2001). Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum. FEMS microbiology letters, 198(1), 57-63.
Martínez, C., Petruccelli, S., Giulietti, A. M., & Alvarez, M. A. (2005). Expression of the antibody 14D9 in Nicotiana tabacum hairy roots. Electronic Journal of Biotechnology, 8(2), 51-57.
Mohanty, A. K., & Wiener, M. C. (2004). Membrane protein expression and production: effects of polyhistidine tag length and position. Protein expression and purification, 33(2), 311-325.
Nezafat, N., Karimi, Z., Eslami, M., Mohkam, M., Zandian, S., & Ghasemi, Y. (2016). Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches. Computational biology and chemistry, 62, 82-95.
Oliveira, C., & Domingues, L. (2018). Guidelines to reach high-quality purified recombinant proteins. Applied microbiology and biotechnology, 102(1), 81-92.
Pham, N. B., Schäfer, H., & Wink, M. (2012). Production and secretion of recombinant thaumatin in tobacco hairy root cultures. Biotechnology journal, 7(4), 537-545.
Poria, V., Rana, A., Kumari, A., Grewal, J., Pranaw, K., & Singh, S. (2021). Current perspectives on chitinolytic enzymes and their agro-industrial applications. Biology, 10(12), 1319.
Roberts, W. K., & Selitrennikoff, C. P. (1988). Plant and bacterial chitinases differ in antifungal activity. Microbiology, 134(1), 169-176.
Sánchez-Ferrer, A., Pérez-Gilabert, M., Núñez, E., Bru, R., & García-Carmona, F. (1994). Triton X-114 phase partitioning in plant protein purification. Journal of Chromatography A, 668(1), 75-83.
Sharp, J. M., & Doran, P. M. (2001). Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnology progress, 17(6), 979-992.
Srivastava, V., Mehrotra, S., & Mishra, S. (2018). Hairy roots: an effective tool of plant biotechnology: Springer.
Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 60(5), 523-533.
Varasteh-Shams, M., Nazarian-Firouzabadi, F., & Ismaili, A. (2020). The direct and indirect transformation methods on expressing a recombinant Dermaseptin peptide in tobacco transgenic hairy root clones. Current Plant Biology, 24, 100177.
Woods, R. R., Geyer, B. C., & Mor, T. S. (2008). Hairy-root organ cultures for the production of human acetylcholinesterase. BMC biotechnology, 8(1), 1-7.
Zayakina, O., Arkhipenko, M., Smirnov, A., Rodionova, N., Karpova, O., & Atabekov, J. (2009). Restoration of potato virus X coat protein capacity for assembly with RNA after His-tag removal. Archives of virology, 154(2), 337-341.
Zhou, J., Chen, J., Zhuang, N., Zhang, A., Chen, K., Xu, N., . . . Jiang, M. (2020). Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1. Frontiers in Bioengineering and Biotechnology, 8, 579.