Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa

Autor(es)

Catani, Cleide Ferreira ^[1] ; Azzoni, Adriano Rodrigues ; Paula, Débora Pires ; Tada, Susely Ferraz Siqueira ^[4] ; Rosselli, Luciana Kauer ^[5] ; Souza, Anete Pereira de ^[6] ; Yano, Tomomasa

Afiliação do(s) autor(es)

^[1] Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia - Brasil

^[4] Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia - Brasil

^[5] Universidade Estadual de Campinas (UNICAMP). COCEN. Centro de Biologia Molecular e Engenharia Genética - Brasil

^[6] Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia - Brasil

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 37, n. 2, p. 320-326, Oct. 2004.
• Área do conhecimento: Ciências Biológicas - Bioquímica
• Assunto(s): Proteínas Vetores de doenças de plantas Bactérias

Resumo

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 °C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy. (AU)

• Processo FAPESP: 01/07533-7 - Resolução da estrutura tridimensional de proteínas relacionadas à patogenicidade de Xyllela fastidiosa
• Beneficiário: Anete Pereira de Souza
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 01/08002-5 - Rede de Biologia Molecular Estrutural - SMolBNet
• Beneficiário: Cleide Ferreira Catani
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado