Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.
Identifieur interne : 000170 ( Main/Exploration ); précédent : 000169; suivant : 000171Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.
Auteurs : Ravindra Bansal [Inde] ; Hiral U. Mistry [Inde] ; Prasun K. Mukherjee [Inde] ; Gagan D. Gupta [Inde]Source :
- Acta crystallographica. Section F, Structural biology communications [ 2053-230X ] ; 2020.
Abstract
Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (VM) of the crystal is 2.32 Å3 Da-1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.
DOI: 10.1107/S2053230X20007025
PubMed: 32510466
PubMed Central: PMC7278501
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Mukherjee</name><affiliation wicri:level="1"><nlm:affiliation>Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085</wicri:regionArea><wicri:noRegion>Mumbai 400085</wicri:noRegion></affiliation></author><author><name sortKey="Gupta, Gagan D" sort="Gupta, Gagan D" uniqKey="Gupta G" first="Gagan D" last="Gupta">Gagan D. Gupta</name><affiliation wicri:level="1"><nlm:affiliation>Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India.</nlm:affiliation><country xml:lang="fr">Inde</country><wicri:regionArea>Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094</wicri:regionArea><wicri:noRegion>Mumbai 400094</wicri:noRegion></affiliation></author></analytic><series><title level="j">Acta crystallographica. Section F, Structural biology communications</title><idno type="eISSN">2053-230X</idno><imprint><date when="2020" type="published">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub> (R<sub>meas</sub> = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V<sub>M</sub>) of the crystal is 2.32 Å<sup>3</sup> Da<sup>-1</sup>, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.</div></front></TEI><pubmed><MedlineCitation Status="In-Process" Owner="NLM"><PMID Version="1">32510466</PMID><DateRevised><Year>2020</Year><Month>10</Month><Day>30</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">2053-230X</ISSN><JournalIssue CitedMedium="Internet"><Volume>76</Volume><Issue>Pt 6</Issue><PubDate><Year>2020</Year><Month>Jun</Month><Day>01</Day></PubDate></JournalIssue><Title>Acta crystallographica. Section F, Structural biology communications</Title><ISOAbbreviation>Acta Crystallogr F Struct Biol Commun</ISOAbbreviation></Journal><ArticleTitle>Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.</ArticleTitle><Pagination><MedlinePgn>257-262</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1107/S2053230X20007025</ELocationID><Abstract><AbstractText>Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub> (R<sub>meas</sub> = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V<sub>M</sub>) of the crystal is 2.32 Å<sup>3</sup> Da<sup>-1</sup>, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Bansal</LastName><ForeName>Ravindra</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mistry</LastName><ForeName>Hiral U</ForeName><Initials>HU</Initials><AffiliationInfo><Affiliation>Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Mukherjee</LastName><ForeName>Prasun K</ForeName><Initials>PK</Initials><AffiliationInfo><Affiliation>Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085, India.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gupta</LastName><ForeName>Gagan D</ForeName><Initials>GD</Initials><AffiliationInfo><Affiliation>Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, India.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>05</Month><Day>29</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Acta Crystallogr F Struct Biol Commun</MedlineTA><NlmUniqueID>101620319</NlmUniqueID><ISSNLinking>2053-230X</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Trichoderma virens</Keyword><Keyword MajorTopicYN="N">effector</Keyword><Keyword MajorTopicYN="N">protein crystallization</Keyword><Keyword MajorTopicYN="N">secreted cysteine-rich 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Gagan D" sort="Gupta, Gagan D" uniqKey="Gupta G" first="Gagan D" last="Gupta">Gagan D. Gupta</name><name sortKey="Mistry, Hiral U" sort="Mistry, Hiral U" uniqKey="Mistry H" first="Hiral U" last="Mistry">Hiral U. Mistry</name><name sortKey="Mukherjee, Prasun K" sort="Mukherjee, Prasun K" uniqKey="Mukherjee P" first="Prasun K" last="Mukherjee">Prasun K. Mukherjee</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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