Production of Abrus pulchellus ribosome-inactivating protein from seeds callus culture.
Ribosome inactivating proteins (RIPs) were isolated from callus culture that were established from seed explants of Abrus pulchellus. Cotyledon segments of 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. A. pulchellus type 2 RIP (pulchellin) expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained after 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins (0.1 M Tris-HCl pH 7.6 buffer, containing 0.15 M NaCl, 3 h at room temperature). A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the presence of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, peak released by 0.1 M galactose, appeared to be composed of two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to that obtained with seeds.
Silva
André Luis C
AL
Grupo de Biofísica Molecular e Espectroscopia, Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, São Carlos, SP CEP 13560-970, Brazil. alcoelho@if.sc.usp.br
Horta
Ana Cecília G
AC
Moreira
Renato A
RA
Beltramini
Leila M
LM
Araújo
Ana Paula U
AP
D029912:Abrus; D000818:Animals; D015153:Blotting, Western; D001938:Brazil; D046508:Culture Techniques; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects*; D006386:Hemagglutination Tests; D007274:Injections, Intraperitoneal; D007928:Lethal Dose 50; D051379:Mice; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000187:drug effects*; D012639:Seeds
D017931:DNA Primers; D010940:Plant Proteins