Pulchellin, a highly toxic type 2 ribosome-inactivating protein from Abrus pulchellus - Cloning, heterologous expression of A-chain and structural studies

• Autor(a) principal: Silva, Andre LC
• Data de Publicação: 2005
• Outros Autores: Goto, Leandro S., Dinarte, Anemari R., Hansen, Daiane [UNIFESP], Moreira, Renanto A., Beltramini, Leila M., Araujo, Ana PU
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNIFESP
• dARK ID: ark:/48912/0013000018294
• DOI: 10.1111/j.1742-4658.2005.04545.x
• Texto Completo: http://dx.doi.org/10.1111/j.1742-4658.2005.04545.x; http://repositorio.unifesp.br/handle/11600/28164
• topic: abrin; lectin; ribosome-inactivating protein; RNA N-glycosidase

Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. the DNA fragment encoding putchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. the deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. the ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. in order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active beterodimer (rPAB). the rPAB showed an apparent molecular mass of approximate to 60 kDa, similar to the native pulchellin. the toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. the rPAB was able to kill 50% of the tested mice with doses of 45 mu g center dot kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. in addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.