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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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

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Author(s):
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
Total Authors: 7
Affiliation:
[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
Total Affiliations: 7
Document type: Journal article
Source: Protein Expression and Purification; v. 37, n. 2, p. 320-326, Oct. 2004.
Field of knowledge: Biological Sciences - Biochemistry
Abstract

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)