A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri
Identifieur interne : 000277 ( Pmc/Checkpoint ); précédent : 000276; suivant : 000278A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri
Auteurs : Giordanni C. Dantas [Brésil] ; Paula M. M. Martins [Brésil] ; Daniela A. B. Martins [Brésil] ; Eleni Gomes [Brésil] ; Henrique Ferreira [Brésil]Source :
- Brazilian Journal of Microbiology [ 1517-8382 ] ; 2016.
Abstract
Citrus canker, caused by the Gram-negative bacterium
Url:
DOI: 10.1016/j.bjm.2016.01.026
PubMed: 26991273
PubMed Central: 4874617
Affiliations:
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A protein expression system for tandem affinity purification in <italic>Xanthomonas citri</italic> subsp. citri</title><author><name sortKey="Dantas, Giordanni C" sort="Dantas, Giordanni C" uniqKey="Dantas G" first="Giordanni C." last="Dantas">Giordanni C. Dantas</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Martins, Paula M M" sort="Martins, Paula M M" uniqKey="Martins P" first="Paula M. M." last="Martins">Paula M. M. Martins</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Martins, Daniela A B" sort="Martins, Daniela A B" uniqKey="Martins D" first="Daniela A. B." last="Martins">Daniela A. B. Martins</name><affiliation wicri:level="2"><nlm:aff id="aff0010">Depto. de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Gomes, Eleni" sort="Gomes, Eleni" uniqKey="Gomes E" first="Eleni" last="Gomes">Eleni Gomes</name><affiliation wicri:level="2"><nlm:aff id="aff0015">Depto. de Biologia, Universidade Estadual Paulista, São Jose do Rio Preto, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Biologia, Universidade Estadual Paulista, São Jose do Rio Preto, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Ferreira, Henrique" sort="Ferreira, Henrique" uniqKey="Ferreira H" first="Henrique" last="Ferreira">Henrique Ferreira</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26991273</idno><idno type="pmc">4874617</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874617</idno><idno type="RBID">PMC:4874617</idno><idno type="doi">10.1016/j.bjm.2016.01.026</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000E65</idno><idno type="wicri:Area/Pmc/Curation">000E64</idno><idno type="wicri:Area/Pmc/Checkpoint">000277</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A protein expression system for tandem affinity purification in <italic>Xanthomonas citri</italic> subsp. citri</title><author><name sortKey="Dantas, Giordanni C" sort="Dantas, Giordanni C" uniqKey="Dantas G" first="Giordanni C." last="Dantas">Giordanni C. Dantas</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Martins, Paula M M" sort="Martins, Paula M M" uniqKey="Martins P" first="Paula M. M." last="Martins">Paula M. M. Martins</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Martins, Daniela A B" sort="Martins, Daniela A B" uniqKey="Martins D" first="Daniela A. B." last="Martins">Daniela A. B. Martins</name><affiliation wicri:level="2"><nlm:aff id="aff0010">Depto. de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Gomes, Eleni" sort="Gomes, Eleni" uniqKey="Gomes E" first="Eleni" last="Gomes">Eleni Gomes</name><affiliation wicri:level="2"><nlm:aff id="aff0015">Depto. de Biologia, Universidade Estadual Paulista, São Jose do Rio Preto, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Biologia, Universidade Estadual Paulista, São Jose do Rio Preto, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author><author><name sortKey="Ferreira, Henrique" sort="Ferreira, Henrique" uniqKey="Ferreira H" first="Henrique" last="Ferreira">Henrique Ferreira</name><affiliation wicri:level="2"><nlm:aff id="aff0005">Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</nlm:aff><country xml:lang="fr">Brésil</country><wicri:regionArea>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP</wicri:regionArea><placeName><region type="state">État de São Paulo</region></placeName></affiliation></author></analytic><series><title level="j">Brazilian Journal of Microbiology</title><idno type="ISSN">1517-8382</idno><idno type="eISSN">1678-4405</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Citrus canker, caused by the Gram-negative bacterium <italic>Xanthomonas citri</italic> subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the <italic>amy</italic> locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium <italic>in vivo</italic>.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Da Silva, A C" uniqKey="Da Silva A">A.C. da Silva</name></author><author><name sortKey="Ferro, J A" uniqKey="Ferro J">J.A. Ferro</name></author><author><name sortKey="Reinach, F C" uniqKey="Reinach F">F.C. Reinach</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alegria, M C" uniqKey="Alegria M">M.C. Alegria</name></author><author><name sortKey="Docena, C" uniqKey="Docena C">C. Docena</name></author><author><name sortKey="Khater, L" uniqKey="Khater L">L. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Braz J Microbiol</journal-id><journal-id journal-id-type="iso-abbrev">Braz. J. Microbiol</journal-id><journal-title-group><journal-title>Brazilian Journal of Microbiology</journal-title></journal-title-group><issn pub-type="ppub">1517-8382</issn><issn pub-type="epub">1678-4405</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26991273</article-id><article-id pub-id-type="pmc">4874617</article-id><article-id pub-id-type="publisher-id">S1517-8382(16)00077-0</article-id><article-id pub-id-type="doi">10.1016/j.bjm.2016.01.026</article-id><article-categories><subj-group subj-group-type="heading"><subject>Genetics and Molecular Microbiology</subject></subj-group></article-categories><title-group><article-title>A protein expression system for tandem affinity purification in <italic>Xanthomonas citri</italic> subsp. citri</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Dantas</surname><given-names>Giordanni C.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Martins</surname><given-names>Paula M.M.</given-names></name><xref rid="aff0005" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author"><name><surname>Martins</surname><given-names>Daniela A.B.</given-names></name><xref rid="aff0010" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Gomes</surname><given-names>Eleni</given-names></name><xref rid="aff0015" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Ferreira</surname><given-names>Henrique</given-names></name><email>henrique.ferreira@linacre.oxon.org</email><xref rid="aff0005" ref-type="aff">a</xref><xref rid="cor0005" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0005"><label>a</label>Depto. de Bioquimica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Rio Claro, SP, Brazil</aff><aff id="aff0010"><label>b</label>Depto. de Bioquímica e Tecnologia Química, Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil</aff><aff id="aff0015"><label>c</label>Depto. de Biologia, Universidade Estadual Paulista, São Jose do Rio Preto, SP, Brazil</aff><author-notes><corresp id="cor0005"><label>⁎</label><italic>Corresponding author</italic>. <email>henrique.ferreira@linacre.oxon.org</email></corresp><fn id="fn1"><label>1</label><p id="npar0005">These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>02</day><month>3</month><year>2016</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><season>Apr-Jun</season><year>2016</year></pub-date><pub-date pub-type="epub"><day>02</day><month>3</month><year>2016</year></pub-date><volume>47</volume><issue>2</issue><fpage>518</fpage><lpage>526</lpage><history><date date-type="received"><day>7</day><month>5</month><year>2015</year></date><date date-type="accepted"><day>23</day><month>10</month><year>2015</year></date></history><permissions><copyright-statement>© 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda.</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>Sociedade Brasileira de Microbiologia</copyright-holder><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/"><license-p>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).</license-p></license></permissions><abstract id="abs0005"><p>Citrus canker, caused by the Gram-negative bacterium <italic>Xanthomonas citri</italic> subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the <italic>amy</italic> locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium <italic>in vivo</italic>.</p></abstract><kwd-group id="kwd0005"><title>Keywords</title><kwd>Citrus canker</kwd><kwd>Expression vectors</kwd><kwd>TAP-tag</kwd></kwd-group></article-meta><notes><p id="misc0005">Associate Editor: Solange Ines Mussatto</p></notes></front><floats-group><fig id="fig0005"><label>Fig. 1</label><caption><p>Expression vectors for Tandem Affinity purification in Xac. (A) Schematic representation of pHF5Ca and pHF5Na: the protein expression vectors for TAP-tagging in Xac. Each vector carries a different TAP-coding DNA, <italic>tap1479</italic> and <italic>tap1761</italic> respectively, which produce TAP fusions to either the C- or N-terminal ends of proteins under study. Both vectors are based on the pCR2.1-TOPO backbone which carries a pUC replication origin and DNA cassettes for ampicillin (Ap) and kanamycin (kan) resistance. (B) DNA sequence of the xylose promoter region common to pHF5Ca and pHF5Na showing the location of the ribosome binding-site (RBS) and the start codon (ATG). (C) The DNA sequences of the 3′-ends of <italic>tap1479</italic> and <italic>tap1761</italic>. Underneath each sequence is the schematic view of the protein fusion that can be produced using the tag. Note that the organization of the modules that compose the TAP-tags (CBP, TEV, and ProtA) differ with respect to TAP1479 and TAP1761 (see text); for the C-terminal fusions, the TAP1479 has ProtA located at the extreme C-terminus of the polypeptide, while ProtA is at the beginning of the protein in the N-terminal fusions. Unique restriction sites for DNA cloning are shown in black; non-unique sites in gray. CBP, calmodulin binding-protein; TEV, Tobacco Etch Virus Protease recognition sequence; ProtA, Protein A from <italic>Staphylococcus aureus</italic>; and EK, enterokinase recognitions sequence (DDDDK, present only in TAP1761); <italic>pxyl</italic>, xylose promoter; <italic>xylR</italic>, the xylose repressor gene; <italic>amy</italic>, an 800 bp fragment of the alpha-amylase gene from Xac.</p></caption><graphic xlink:href="gr1"></graphic><graphic xlink:href="gr1b"></graphic></fig><fig id="fig0010"><label>Fig. 2</label><caption><p>Colony PCR to detect the presence of pHF5Ca integrated into the chromosomal DNA of Xac. Subsequent to electrotransformation of Xac with the expression vector pHF5Ca, six kan<sup>R</sup> mutants were selected and subjected to colony PCR using the primers PBS1479F/R specific for the amplification of the <italic>tap1479</italic> (approximately 640 bp). The PCR amplicons were visualized after migration through a 0.7% agarose gel. Lane: 1, WT Xac (negative control); 2–7, the six selected mutants; 8, PCR using pHF5Ca as the DNA template (positive control); and 9, DNA molecular weight marker (1 kb DNA ladder, NEB).</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig0015"><label>Fig. 3</label><caption><p>The integration of the TAP expression system into the chromosome of Xac does not alter its pathogenicity. Two independently selected mutants of Xac <italic>amy</italic>::pHF5Ca (1 and 2) were inoculated into leaves of sweet orange Bahia alongside the wild type strain (Xac wt) and a sample of NYG-medium (negative control). A map of the inoculation is shown on the right-hand side of the figure. Pictures were taken immediately after inoculation (A–C) and also 7 days post-inoculation (D–F). The test was conducted in triplicate (A–C).</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="fig0025"><label>Fig. 4</label><caption><p>Western blotting analysis to detect expression of TAP1479 by Xac <italic>amy</italic>::pHF5Ca mutants. The functionality of the TAP expression system was verified by the ability of an <italic>E. coli</italic> DH10B strain, with pHF5Ca and Xac kan<sup>R</sup> mutants, carrying the vector integrated into the chromosome, to produce the TAP1479 (approximately 21 kDa) (details of the induction procedure have been described in “Materials and methods” section). (A) Coomassie blue-stained 10% SDS-PAGE showing the separation of proteins from cell extracts of <italic>E. coli</italic> and Xac; lanes: 1, <italic>E. coli</italic>/pHF5Ca; 2, <italic>E. coli</italic>/pHF5Ca + 0.5% xylose; 3, Xac (WT); 4, Xac WT + 0.5% xylose; 5, Xac <italic>amy</italic>::pHF5Ca mutant 1; 6, Xac <italic>amy</italic>::pHF5Ca mutant 1 + 0.5% xylose; 7, Xac <italic>amy</italic>::pHF5Ca mutant 2, 8; Xac <italic>amy</italic>::pHF5Ca mutant 2 + 0.5% xylose; and 9, protein molecular weight marker. (B) X-ray film displaying bands detected by the Western blotting analysis. Proteins in a replica of the gel shown in A were transferred to a PVDF membrane and exposed to a secondary antibody coupled to HRP. Lanes are the same as in (A). The position of the TAP1479 is marked with a black arrow on the right hand side of the film.</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="fig0020"><label>Fig. 5</label><caption><p>Purification of TAP1479 from <italic>E. coli</italic>/pHF5Ca and Xac <italic>amy</italic>::pHF5Ca. <italic>E. coli</italic> DH10B carrying pHF5Ca and a kan<sup>R</sup> mutant of Xac harboring pHF5Ca integrated into its chromosome were induced for the production of the TAP1479. Following expression, cells were lysed and protein extracts subjected to affinity chromatography through IgG-sepharose. (A) <italic>E. coli</italic>/pHF5Ca; and (B) Xac <italic>amy</italic>::pHF5Ca, Coomassie blue-stained 10% SDS-PAGE showing the separation of proteins from the fractions collect throughout the process. (A) lane: 1, clarified lysate; 2, flow through; 3–6, washes (20 column-volumes each); 7, protein molecular weight marker; 8–14, elution fractions. (B) Lane: 1, clarified lysate; 2, flow through; 3–5, washes; 6, protein molecular weight marker; 7–14, elution fractions. The position of the TAP1479 is marked with black arrows in both gels.</p></caption><graphic xlink:href="gr5"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Brésil</li></country><region><li>État de São Paulo</li></region></list><tree><country name="Brésil"><region name="État de São Paulo"><name sortKey="Dantas, Giordanni C" sort="Dantas, Giordanni C" uniqKey="Dantas G" first="Giordanni C." last="Dantas">Giordanni C. Dantas</name></region><name sortKey="Ferreira, Henrique" sort="Ferreira, Henrique" uniqKey="Ferreira H" first="Henrique" last="Ferreira">Henrique Ferreira</name><name sortKey="Gomes, Eleni" sort="Gomes, Eleni" uniqKey="Gomes E" first="Eleni" last="Gomes">Eleni Gomes</name><name sortKey="Martins, Daniela A B" sort="Martins, Daniela A B" uniqKey="Martins D" first="Daniela A. B." last="Martins">Daniela A. B. Martins</name><name sortKey="Martins, Paula M M" sort="Martins, Paula M M" uniqKey="Martins P" first="Paula M. M." last="Martins">Paula M. M. Martins</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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