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Master's Dissertation
DOI
10.11606/D.87.2008.tde-07012009-121801
Document
Author
Full name
Fabiano Tófoli de Araújo
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Fernandes, Andrea Balan (President)
Baldini, Regina Lúcia
Vicente, Elisabete Jose
Title in Portuguese
Clonagem, expressão e purificação da proteína ligadora de alcano sulfonatos do sistema de transporte ABC de Xanthomonas axonopodis pv.citri.
Keywords in Portuguese
Xanthomonas
Cristalografia
Cromatografia afinidade
Dicroísmo circular
Fluorimetria
Proteínas de transporte
Abstract in Portuguese
O genoma de Xanthomonas citri (Xac) possui mais de 20 tipos de transportadores do tipo ABC incluindo o operon ssuABC associado ao transporte de alcano sulfonatos. Deste operon, escolhemos a proteína periplasmática ligadora de alcano sulfonatos SsuA2, para caracterização e análises espectroscópicas e estruturais. A rSsuA2 foi expressa no citoplasma de células de Escherichia coli e utilizada para a preparação de anticorpos em camundongos, que foram capazes de reconhecer a proteína recombinante, mas não a nativa no extrato de células de Xac. A rSssuA2 apresenta estrutura característica de proteínas alfa-beta, maior estabilidade em pH neutro (7.0), como também foi evidenciado pela obtenção de cristais somente nesta faixa, e pouca flexibilidade ao desenovelamento térmico. Os cristais difratam com resolução de 1.8 Å e pertencem ao grupo espacial de simetria P21. Além do o operon ssu (ssu2) altamente conservado, Xac apresenta o operon tau (ssu1) para captação de taurina. O papel do operon para Xac é discutido.
Title in English
Cloning, expression and purification of the Xanthomonas axonopodis pv citri ABC transport alkanosulphonate-binding protein.
Keywords in English
Xanthomonas
Afinity cromatography
Crystallography
Dichroism circular
Fluorimetry
Transport proteins
Abstract in English
Xanthomonas citri (Xac) genome has more than 20 different ABC transporters, including the ssuABC operon. In this work, the alkanosulphonate-periplasmic binding protein SsuA2 was chosen for spectroscopic and structural analysis. The rSsuA2 protein was expressed as a soluble form and purified by immobilized metal affinity chromatography. Antibodies produced from the recombinant protein were able to recognize the rSsuA2, but not the native protein in the Xac extract samples. The protein presents secondary structure defined by alfa helices and beta-sheets, high stability in neutral pH and low flexibility to the thermal denaturation. The determination of the optimal pH range was important to produce crystals of high quality diffracting at 1.8 Å with symmetry of the P21 spatial group. Besides the highly conserved operon ssu (ssu2), Xac has the tau operon (ssu1) for taurine uptake.
 
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Publishing Date
2009-02-17
 
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