PRODUCTION OF A RIBOSOME-INACTIVATING PROTEIN FROM CALLUS CULTURE OF Abrus pulchellus. PRELIMINARY STUDIES

Silva, A.L.C.(1), Horta, A.C.G. (2), Moreira, R.A. (2), Beltramini, L. M. (1), Araújo, A.P.U.(1)

(1)Grupo de Biofísica Molecular e Espectroscopia, IFSC, USP, (2)Laboratório de Lectinas e Glicoconjugados, Depto. de Bioquímica e Biologia Molecular, UFC.

Plant ribosome-inactivating proteins (RIPs) are RNA–N-glycosidases that cleave a specific ribosomal RNA site preventing the normal protein synthesis. Pulchelinis a Type 2 RIPs, like ricin and abrin, consisting on a lectin specific galactose-binding subunit, which recognizes sites on the surface of a target-cell, and one ribosome-inactivating N-glicosidase subunit. These proteins form a class of highly toxic molecules which specifically kill cell by their N-glycosidase enzymatic activity under the 60S ribosomal subunit. Although their potency to kill cells is remarkable, the RIPs have been explored in the chemical construction of drugs to be used as “biological missile” in the clinical treatment of human diseases. With the aim for investigate the production of pulchelin in callus culture, cotyledon segments of Abrus pulchellus immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin (2,4-D), citokinin (kinetin and BA) and sucrose in order to determine the best callus induction. The explants were maintained in a dark growth room at 28 + 2^oC. The presence of the mRNA from Pulchelin was observed in callus by RT-PCR technique. The calli obtained after 35 days period were freeze dried, macerated and submitted to protein extraction with buffers of different pH values (2.6, 4.0, 6.0, 7.6 and 10.0) and the proteins concentration in the extracts was determined by Bradford method. The pH 7.6 and 10 buffers were the most efficient to extract the largest amount of protein. The calli crude extract (pH 7.6) showed hemagglutinating activity against rabbit blood cells and showed a high toxicity to mice when administered intraperitoneally. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein peak released by 0.1M D-galactose showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to results obtained with seeds.

Supported by CNPq

CORRESPONDENCE TO:

ANDRÉ LUIS COELHO DA SILVA, RUA JOÃO DE OLIVEIRA JR., No. 50, Apto.501, SÃO CARLOS, SP, CEP: 13560-970, Brasil, Email: alcoelho@if.sc.usp.br