THE OPTIMISED CONDITIONS OF INDUCTION OF RECOMBINANT RIP rMJC15310 ACTIVITY ISOLATED FROM Mirabilis jalapa L. LEAVES
Ribosome Inactivating Proteins (RIPs) are compounds isolated from plants with ability to inhibit protein synthesis. The inhibition of protein synthesis is due to inactivation of ribosomal RNA through a site-specific deadenylation mediated by RNA N-glycosidase. Reportedly, RIPs mainly possess wide range of bioactivity including antiviral activity against plant infections. Other activities of RIP were as abortifacien, antivirus and anticancer. This study was aimed to isolate and characterize the optimum conditions for inducing the expression of recombinant RIPs isolated from the leaves of Mirabilis Jalapa L. We have been successfully isolated several RIPs and engineered these proteins to be expressed in E. coli. These recombinant proteins were obtained by screening cDNA library originated from the mRNA of Mirabilis jalapa L leaves, and inserted into pUC19 carrying lacZ gene. The presence of recombinant plasmid was tested by using α-complementation assay. Many RIPs have been isolated from plants and these proteins express enzymatic activity by cutting supercoiled double stranded DNA. One RIP namely rMJC15310 was obtained from this study and the proteins having ~ 8kb in size, cut the supercoiled DNA into linear form at the concentration as low as 5 µg. The ability to cut supercoiled DNA increased on inducing its expression with 0.4% IPTG.
Key words: Ribosome Inactivating Proteins (RIP), IPTG, Mirabilis jalapa L., recombinant protein
Barbieri, L., Batteli, M.A., Stirpe, F., 1993, Ribosome inactivating proteins from Plants, Biochem et Biophys. Acta, 405-410
Barbieri, L., Valbonesi, P., Bonora, E., Gorini, P., Bolognesi, A., and Stirpe, F., 1997, Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins : effect on DNA, RNA and poly(A). Nucleic Acids Res. 25, 518–522
Barberi, L., Polito, L., Bolognesi, A., Ciani, M., Pelosi, E., Farini, V., Jha, A.K., Sharma,N., Vivano, J.M., Chambery, A., Parente, A., and Stirpe, F., 2006,
Ribosomeinactiva-ting proteins in edible plants and purification and characterization of a new ribosomeinactivating protein from Cucurbita moschata, Biochim. Biophys. Acta, 170: 783-792.
Batteli, M.G., 2004, Cytotoxicity and toxicity to animals and humans of ribosomeinactivating proteins. Mini Rev Med Chem, 4(5):513-21.
Endo, Y., and Tsurugi, K.,1987, RNA Nglycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128–8130
Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K., 1987, The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J. Biol. Chem. 262, 5908–5912
Li, M.X., Yeung, H.-W., Pan, L.P. and Chan, S.I.,1991, Nucleic Acids Res. 19, 63096312.
Lin, J. Y., Tserng, K. Y., Chen, C. C., Lin, L. T., and Tung, T. C., 1970, Abrin and ricin: new anti-tumour substances. Nature (London) 227, 292–293
Ling, J., Lui, W., and Wang, T.P., 1994, Cleavage of supercoiled double stranded DNA by several ribosome inactivating proteins in vitro, FEBS Letters, 345: 143-146.
Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell., J.E., 2000, Molecular Cell Biology, 4th Ed., 244, W.H. Freeman and Company, New York.
Narayanan, S., K. Surendranath, N. Bora, A. Surolia, and A.A. Karande. 2005. Ribosome inactivating proteins and apoptosis. FEBS Lett. 579:1324-1331.
Peumans,W., Hoa,Q., and van Damme,E., 2001, Ribosome-inactivating proteins from plants: more than RNA Nglycosidase? J FASEB, 15: 1493-1506
Qu,X. and Qing,L., 2004, Abrin induces cell apoptosis by cytochrom c release and caspaseactivation,J.Bio.Mic.37(4),445-453.
Roulean, C., Curiel, M., Weber, W., Smale, R., Kurtzberg, L., Masearello, J., Berger, C., Waller, G., Bagley, R., Honma, N., Hasegawa,K., Ishida,I., Kataoka,S., Mehraenin,K., Horten, Miller,G., and Teicher,B.H., 2008, Endosialin protein expression and therapeutic target potential in human solid tumors: Sarcoma versus carcinoma, Clin. Can.Res.14, 7223.
Sambrook,J., Fritsch,E.F., Maniatis,T., 1989, Molecular Cloning : a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York.
Sha, O., Yew, D.T., Ng, T.B., Yuan, L., Kwong, W.H., 2010, Different in vitro toxicities of structurally similar type I ribosomeinactivating proteins (RIPs). Toxicol In Vitro. 24 (4):1176-82
Sismindari, Husaana,A., Mubarika,S., 1998, In vitro cleavage of Supercoiled DNA by Annona squamosa extract, Indonesian J. Pharm., 9 (4), 146-152.
Sismindari, Mubarika S, and Sudjadi, 2001, Selective cytotoxic effects on cancer celllines of protein fraction isolated from Annona squamosa and containing ribosome-inactivating proteins, J.Biotech. 459 – 462.
Sismindari, Handayani A., Yulia S., and Chandra E., 2002, Potent effects of protein extract isolated from Erythrina fusca Lour leaves on cancer cell-lines,. J.Biotech. 559-564
Stirpe, F., Barbieri, R., Battteli, MG., Soria, M., and Iappi, D.A., 1992, RIPs from Plants: Present status and Future prospect, Rev.Biotech 10: 105-109
Stirpe,F., 2004, Ribosome-inactivating proteins, Toxicon, 44, 371 – 383
Sudjadi, Sismindari, Astuti,P., 2010, Kloning gena RIP penyandi Ribosome Inactivating protein dari daun Mirabilis jalapa L dengan pendekatan cDNA library, Proceeding Kongres Ilmiah IAI XVIII dan Rapat Kerja Nasional 2010, Makasar.
Wang, P., Zoubenko, O., and Tumer, N. E.,1998, Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II. Plant Mol. Biol. 38, 957–964
- There are currently no refbacks.
Copyright (c) 2017 INDONESIAN JOURNAL OF PHARMACY
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
Indonesian J Pharm indexed by:
View My Stats