Purificação e caracterização biológica da lectina recombinante de Canavalia brasiliensis (rConBr) produzida em Escherichia coli

• Ano de defesa: 1999
• Autor(a) principal: Nogueira, Nádia Accioly Pinto
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Biologia molecular; Proteína recombinante; Escherichia coli; Canavalia brasiliensis
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/44565

ConBr is a D-glucose/D-mannose binding lectinis isolated from Canavalia brasiliensis seeds. Like ConA, ConB rissynthesized during seed development as a pre-prolectin which undergoes a complex series of pos-translational events to produce the mature lectin. The pre-pro-ConBr was expressed in E. coli using the pET15b expression vector. The recombinant lectin (rConBr) was expressed by growing the bacteria in the presence of isopropyl B-D-thiogalactopyranoside. AlI the recombinant lectin was found in an insoluble aggregated form as inclusion bodies. After solubilization of the inclusion bodies with 6M guanidine hydrochloride, rConBr was purified to homogeneity under denaturing conditions using Ni2+ affinity chromatography. The recombinant lectin, obtained at ayield of 15 - 30 mg/l culture, had a higher molecular mass (37 kDa) than the native lectin (30 kDa) as estimated by SDS-PAGE and Western blot analyses, showing that the pre-pro-ConBr is not processed in E. coli cells. The identity of the expressed protein as the unprocessed pre-pro-ConBr was confirmed by N-terminal protein sequencing. The recombinant lectin was able to bind carbohydrates, to agglutinate rabbit erythrocytes and to induce pro-inflammatory effects in rats and histamine release from rat mast cells. However, rConBr was far less potent than the native lectin regarding its biological activities. Therefore, the complex posttranslational events that takes place in the plant during ConBr biosynthesis seem to be necessary to produce a thoroughly active lectin.