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

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

Autor Silva, Andre LC Google Scholar
Goto, Leandro S. Google Scholar
Dinarte, Anemari R. Google Scholar
Hansen, Daiane Autor UNIFESP Google Scholar
Moreira, Renanto A. Google Scholar
Beltramini, Leila M. Google Scholar
Araujo, Ana PU Google Scholar
Instituição Universidade de São Paulo (USP)
Fundacao Hemoctr Ribeirao Preto
Universidade Federal de São Paulo (UNIFESP)
Univ Fed Ceara
Resumo 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.
Palavra-chave abrin
lectin
ribosome-inactivating protein
RNA N-glycosidase
Idioma Inglês
Data de publicação 2005-03-01
Publicado em Febs Journal. Oxford: Blackwell Publishing Ltd, v. 272, n. 5, p. 1201-1210, 2005.
ISSN 1742-464X (Sherpa/Romeo, fator de impacto)
Publicador Blackwell Publishing Ltd
Extensão 1201-1210
Fonte http://dx.doi.org/10.1111/j.1742-4658.2005.04545.x
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000227497500012
Endereço permanente http://repositorio.unifesp.br/handle/11600/28164

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