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

被引：8

作者：

Catani, CF

Azzoni, AR

Paula, DP

Tada, SFS

Rosselli, LK

de Souza, AP

Yano, T

机构：

[1] Univ Estadual Campinas, Inst Biol, Dept Microbiol & Immunol, Campinas, SP, Brazil

[2] Univ Estadual Campinas, Inst Biol, Genet Engn & Mol Biol Ctr, Dept Genet & Evolut, BR-13083970 Campinas, SP, Brazil

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2004年 / 37卷 / 02期

基金：

巴西圣保罗研究基金会;

关键词：

VapD protein; expression and purification; Xylella fastidiosa;

D O I：

10.1016/j.pep.2004.07.002

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 37degreesC. 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. (C) 2004 Elsevier Inc. All rights reserved.

引用

页码：320 / 326

页数：7

共 32 条

[1]

BARETTA MJG, 1992, P INT SOC, V2, P830

[2] Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response [J].

Benoit, S ;

Benachour, A ;

Taouji, S ;

Auffray, Y ;

Hartke, A .

RESEARCH IN MICROBIOLOGY, 2001, 152 (05) :439-449

[3] Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights [J].

Bhattacharyya, A ;

Stilwagen, S ;

Reznik, G ;

Feil, H ;

Feil, WS ;

Anderson, I ;

Bernal, A ;

D'Souza, M ;

Ivanova, N ;

Kapatral, V ;

Larsen, N ;

Los, T ;

Lykidis, A ;

Selkov, E ;

Walunas, TL ;

Purcell, A ;

Edwards, RA ;

Hawkins, T ;

Haselkorn, R ;

Overbeek, R ;

Kyrpides, NC ;

Predki, PF .

GENOME RESEARCH, 2002, 12 (10) :1556-1563

[4]

BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3

[5] High-level genetic diversity in the vapD chromosomal region of Helicobacter pylori [J].

Cao, P ;

Cover, TL .

JOURNAL OF BACTERIOLOGY, 1997, 179 (09) :2852-2856

[6] Comparison of the genomes of two Xanthomonas pathogens with differing host specificities [J].

A. C. R. da Silva ;

J. A. Ferro ;

F. C. Reinach ;

C. S. Farah ;

L. R. Furlan ;

R. B. Quaggio ;

C. B. Monteiro-Vitorello ;

M. A. Van Sluys ;

N. F. Almeida ;

L. M. C. Alves ;

A. M. do Amaral ;

M. C. Bertolini ;

L. E. A. Camargo ;

G. Camarotte ;

F. Cannavan ;

J. Cardozo ;

F. Chambergo ;

L. P. Ciapina ;

R. M. B. Cicarelli ;

L. L. Coutinho ;

J. R. Cursino-Santos ;

H. El-Dorry ;

J. B. Faria ;

A. J. S. Ferreira ;

R. C. C. Ferreira ;

M. I. T. Ferro ;

E. F. Formighieri ;

M. C. Franco ;

C. C. Greggio ;

A. Gruber ;

A. M. Katsuyama ;

L. T. Kishi ;

R. P. Leite ;

E. G. M. Lemos ;

M. V. F. Lemos ;

E. C. Locali ;

M. A. Machado ;

A. M. B. N. Madeira ;

N. M. Martinez-Rossi ;

E. C. Martins ;

J. Meidanis ;

C. F. M. Menck ;

C. Y. Miyaki ;

D. H. Moon ;

L. M. Moreira ;

M. T. M. Novo ;

V. K. Okura ;

M. C. Oliveira ;

V. R. Oliveira ;

H. A. Pereira .

Nature, 2002, 417 (6887) :459-463

[7]

Dow JM, 2000, YEAST, V17, P263, DOI 10.1002/1097-0061(200012)17:4&lt

[8]

263::AID-YEA44&gt

[9]

3.0.CO

[10]

2-G

← 1 2 3 4 →