Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3

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dc.contributor.authorCHURA-CHAMBI, ROSA M.
dc.contributor.authorFRAGA, TATIANA R.
dc.contributor.authorSILVA, LUDMILA B. da
dc.contributor.authorYAMAMOTO, BRUNO B.
dc.contributor.authorISAAC, LOURDES
dc.contributor.authorBARBOSA, ANGELA S.
dc.contributor.authorMORGANTI, LIGIA
dc.coverageInternacionalpt_BR
dc.date.accessioned2018-11-29T16:44:58Z
dc.date.available2018-11-29T16:44:58Z
dc.date.issued2018pt_BR
dc.description.abstractEnzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.pt_BR
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)pt_BR
dc.description.sponsorshipIDFAPESP: 15/02574-0; 14/00926-3; 13/ 17419-4; 16/17534-6pt_BR
dc.format.extent1-7pt_BR
dc.identifier.citationCHURA-CHAMBI, ROSA M.; FRAGA, TATIANA R.; SILVA, LUDMILA B. da; YAMAMOTO, BRUNO B.; ISAAC, LOURDES; BARBOSA, ANGELA S.; MORGANTI, LIGIA. Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3. <b>Biotechnology Reports</b>, v. 19, n. e00266, p. 1-7, 2018. DOI: <a href="https://dx.doi.org/10.1016/j.btre.2018.e00266">10.1016/j.btre.2018.e00266</a>. Disponível em: http://repositorio.ipen.br/handle/123456789/29292.
dc.identifier.doi10.1016/j.btre.2018.e00266pt_BR
dc.identifier.fasciculoe00266pt_BR
dc.identifier.issn2215-017Xpt_BR
dc.identifier.orcidaguardandopt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-7870-1793
dc.identifier.percentilfiSem Percentil
dc.identifier.percentilfiCiteScore81.50
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/29292
dc.identifier.vol19pt_BR
dc.relation.ispartofBiotechnology Reportspt_BR
dc.rightsopenAccesspt_BR
dc.subjectbacteria
dc.subjectenzymes
dc.subjectmetalloproteins
dc.subjectfluorescence
dc.subjectlysine
dc.subjectproteins
dc.subjecthydrostatics
dc.subjectpressure dependence
dc.titleLeptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3pt_BR
dc.typeArtigo de periódicopt_BR
dspace.entity.typePublication
ipen.autorLIGIA ELY MORGANTI FERREIRA DIAS
ipen.autorROSA MARIA CHURA CHAMBI
ipen.codigoautor296
ipen.codigoautor3338
ipen.contributor.ipenauthorLIGIA ELY MORGANTI FERREIRA DIAS
ipen.contributor.ipenauthorROSA MARIA CHURA CHAMBI
ipen.date.recebimento18-11pt_BR
ipen.identifier.fiSem F.I.pt_BR
ipen.identifier.fiCiteScore6.4
ipen.identifier.ipendoc25079pt_BR
ipen.type.genreArtigo
relation.isAuthorOfPublicationc26a78ae-be8e-4a8d-b22c-0a62eb422f8b
relation.isAuthorOfPublicationd2b97487-e004-4056-8df7-0ab5c8d5cacc
relation.isAuthorOfPublication.latestForDiscoveryd2b97487-e004-4056-8df7-0ab5c8d5cacc
sigepi.autor.atividadeCHURA-CHAMBI, ROSA M.:3338:820:Spt_BR
sigepi.autor.atividadeMORGANTI, LIGIA:296:820:Npt_BR
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