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http://hdl.handle.net/10261/158203
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dc.contributor.author | Sonderegger, Christoph | es_ES |
dc.contributor.author | Galgóczy, László | es_ES |
dc.contributor.author | Garrigues, Sandra | es_ES |
dc.contributor.author | Fizil, Adam | es_ES |
dc.contributor.author | Borics, Attila | es_ES |
dc.contributor.author | Manzanares, Paloma | es_ES |
dc.contributor.author | Hegedues, Nikoletta | es_ES |
dc.contributor.author | Huber, Anna | es_ES |
dc.contributor.author | Marcos López, José Francisco | es_ES |
dc.contributor.author | Batta, Gyula | es_ES |
dc.contributor.author | Marx, Florentine | es_ES |
dc.date.accessioned | 2017-12-15T07:47:54Z | - |
dc.date.available | 2017-12-15T07:47:54Z | - |
dc.date.issued | 2016-11-11 | - |
dc.identifier.citation | Microbial Cell Factories 15: 192 (2016) | es_ES |
dc.identifier.uri | http://hdl.handle.net/10261/158203 | - |
dc.description.abstract | Background: Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses. Results: The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays. Conclusion: This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses. | es_ES |
dc.description.sponsorship | This study was supported by the Austrian Science Fund (FWF P25894-B20 to FM, FWF I1644-B20 to FM), the Hungarian Science Fund (OTKA ANN 110821 to GB), and the Spanish Ministry of Economy (BIO2015-68790-C2-1-R MINECO/FEDER to PM and JFM). LG holds a Lise Meitner fellowship from the Austrian Science Fund FWF (M1776-B20). SG holds a FPU doctoral fellowship (MECD FPU13/04584) from the Spanish Ministry of Education. The research of AB was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. | es_ES |
dc.language.iso | eng | es_ES |
dc.publisher | BioMed Central | es_ES |
dc.relation | info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2015-68790-C2-1-R | es_ES |
dc.relation.isversionof | Publisher's version | es_ES |
dc.rights | openAccess | es_ES |
dc.subject | Antifungal proteins | es_ES |
dc.subject | PAF | es_ES |
dc.subject | NFAP | es_ES |
dc.subject | Penicillium chrysogenum | es_ES |
dc.subject | Penicillium digitatum | es_ES |
dc.subject | Neosartorya fischeri | es_ES |
dc.subject | Recombinant protein production | es_ES |
dc.subject | Electronic circular dichroism (ECD) spectroscopy | es_ES |
dc.subject | Nuclear magnetic resonance (NMR) | es_ES |
dc.title | A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses | es_ES |
dc.type | artículo | es_ES |
dc.identifier.doi | 10.1186/s12934-016-0586-4 | - |
dc.description.peerreviewed | Peer reviewed | es_ES |
dc.relation.publisherversion | https://doi.org/10.1186/s12934-016-0586-4 | es_ES |
dc.identifier.e-issn | 1475-2859 | - |
dc.rights.license | http://creativecommons.org/licenses/by/4.0/ | es_ES |
dc.contributor.funder | Austrian Science Fund | es_ES |
dc.contributor.funder | Hungarian Scientific Research Fund | es_ES |
dc.contributor.funder | Ministerio de Economía y Competitividad (España) | es_ES |
dc.contributor.funder | European Commission | es_ES |
dc.relation.csic | Sí | es_ES |
oprm.item.hasRevision | no ko 0 false | * |
dc.identifier.funder | http://dx.doi.org/10.13039/501100003329 | es_ES |
dc.identifier.funder | http://dx.doi.org/10.13039/501100002428 | es_ES |
dc.identifier.funder | http://dx.doi.org/10.13039/501100000780 | es_ES |
dc.identifier.pmid | 27835989 | - |
dc.type.coar | http://purl.org/coar/resource_type/c_6501 | es_ES |
item.openairetype | artículo | - |
item.grantfulltext | open | - |
item.cerifentitytype | Publications | - |
item.openairecristype | http://purl.org/coar/resource_type/c_18cf | - |
item.fulltext | With Fulltext | - |
item.languageiso639-1 | en | - |
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