PURIFICATION OF RIBOSOME-INACTIVATING PROTEIN (RIP) OF MIRABILIS JALAPA L. LEAVES BY CM-SEPHAROSE CL-6B AND SEPHACRYL S-300HR COLUMN

Sudjadi ., Sismindari ., Tenti Herawati, Alberta Tri Prasetyowati

Abstract


Total protein of Mirabilis jalapa leaves has activities to cleave supercoiled DNA to nick circular and linear and to cleave glycosidic bound of adenin4324 of yeast 26S rRNA. The protein was cytotoxic on HeLa and Raji celllines through apoptosis and necrosis mechanisms, respectively. However, the protein, poseess the activities, has yet been resolved. Therefore, protein furification to obtain single protein is necessary. Crude extract of M.jalapa leaves was prepared using 5 mM sodium phosphate buffer pH 7,2 containing 0,14 M sodium chloride. Total protein was obtained by precipitating the extract at 100% saturated ammonium sulfate and then dialyzed against phosphate buffer. The protein was purified by CM-Sepharose CL-6B, a cation exchange column. After loading the protein, the column was washed with 5 mM sodium phosphate buffer pH 6,5. The proteins were then eluted with linear gradient of increasing sodium chloride concentration. The fraction with supercoiled DNA-cutting activity was performed for N-glycosidase activity. The active fraction was a subject for further purification with Sephacryl S-300HR, a gel filtration column. The purity and size protein were confirmed by SDS-polyacrylamide gel electrophoresis with silver nitrate staining. RIP like protein was eluted from CM-Sepharose CL-6B on 0,25 – 0,3 M sodium chloride. The size of protein is around 30 kD.

Key words : RIP purification, M.jalapa leaves, CM-Sepharose CL-6B, Sephacryl S-300HR


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References


Chaddock, J.A., Mozingo,A.E., Robertus,J.D. Lord.J.M., and Roberts.L.M., 1996, Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity, J.Biochem., 235, 159-166.

Ikawati,Z, Sudjadi, Sismindari, Sari,R.P. dan Maulani,N., 2002, Efek fraksi protein sejenis RIP (Ribosominactivating protein) yang diisolasi dari akar Mirabilis jalapa L terhadap proses kematian sel Raji, Biologi, 2, 769-783.

Ling, J., Liu, W., Wang, T.P., 1994, Cleavage of supercoiled double stranded DNA by several ribosome inactivating proteins in vitro, FEBS Letters, 345, 143-146

May, M.J., Hartley, M.R., Roberts, L.M., Kreig, P.A., Osborn, R.W., and Lord, J.M., 1989, Ribosome inactivation by ricin A-chain: a sensitive method to assess the activity of wild-type and mutant polypeptides, EMBO J., 8, 301-308.

Roncuzzi.L. and Gasperi-Campani,A., 1996, DNA nuclease activity of the single chain ribosome inactivating proteins dianthin 30, saporin 6 and gelonin, FEBS Lett, 392, 16 – 20.

Sambrook.J., Fritsch,,E.F. and Maniatis,T., 1989, Molecular cloning, A laboratory manual, Second edition, Cold Spring Harbor Laboratory Press, New York.

Sismindari and Lord,J.M., 2000, Ribosome Inactivating Protein: RNA N-glycosidase activity of Mirabilis jalapa L, Morinda citrifolia L and Carica papaya L, Indon J.Biotech, 324-345.

Stirpe,F. and Barbieri,L., 1986, Ribosom-inactivating proteins up to date, FEBS Lett, 195, 1-8.

Stirpe, F., Barbieri, R., Batteli, M.G., Soria, M., Lappi, D.A., 1992, RIP from Plants: Present status and future prospect, Review, Biotechnology, 10, 105-109.

Sudjadi, Ikawati,Z., Sismindari, Krisandika,F.A., 2002, Efek fraksi protein dari biji Mirabilis jalapa L pada proses kematian sel HeLa, Biologi, 2, 743-754.

Suprapti,E., 1997, Identifikasi RIPs dari daun akar dan biji Kembang Pukul Empat (Mirabilis jalapa L) yang tumbuh di Jawa, Indonesia, Skripsi, Fakultas Farmasi UGM, Yogyakarta.

Vivanco, J.M., Qurci, M., Salazar, L.F., 1999a, Antiviral and antiviroid activity of MAP-containing extracts from Mirabilis jalapa roots. Plant Dis. 83, 1116-1121.

Vivanco, J.M., Sovary, B.J. and Flores, H.E., 1999b, Characterization of two novel typeI Tibosome-Inactivating Proteins from the sorage roots of Mirabilis expansa, Plant Physiol., 1447-1456




DOI: http://dx.doi.org/10.14499/indonesianjpharm0iss0pp316-321

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