Expression of the human prolactin antagonist delta 1-11 G129R-PRL in E. coli periplasm

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dc.contributor.authorSUZUKI, M.F.pt_BR
dc.contributor.authorALMEIDA, L.A.pt_BR
dc.contributor.authorPOMIN, S.A.pt_BR
dc.contributor.authorSILVA, F.D.pt_BR
dc.contributor.authorFREIRE, R.P.pt_BR
dc.contributor.authorOLIVEIRA, J.E.pt_BR
dc.contributor.authorAFFONSO, R.pt_BR
dc.contributor.authorBARTOLINI, P.pt_BR
dc.contributor.authorSOARES, C.R.J.pt_BR
dc.contributor.editorSANTOS, NATHALIA V. dospt_BR
dc.coverageInternacionalpt_BR
dc.creator.eventoBIOPARTITIONING & PURIFICATION CONFERENCEpt_BR
dc.date.accessioned2020-03-26T16:58:37Z
dc.date.available2020-03-26T16:58:37Z
dc.date.eventoNovember 11-13, 2019pt_BR
dc.description.abstractRecombinant human prolactin antagonist delta 1-11 G129R-hPRL is a 21.9 kDa protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining an authentic, soluble, and correctly folded protein, as an alternative to the cytoplasmic production in inclusion bodies of an unfolded, insoluble protein, carrying an extra initial methionine. The aim of this work was to carry out the expression of delta 1-11 G129R-hPRL antagonist in the periplasm of E. coli, testing different temperatures. E. coli BL21(DE) strain, transformed with a plasmid based on a pET25b(+) vector, DsbA signal peptide and delta 1-11 G129R-hPRL cDNA, was cultured in LB medium with ampicillin addition. After overnight culture at 30 °C, 0.6 mM IPTG was added and five different temperatures were applied: 25, 30, 32, 35 and 37 °C. Periplasmic fluid was extracted after 5 hours by osmotic shock. The samples were analyzed by SDS-PAGE, Western blotting and RP-HPLC. The best condition was increasing the temperature to 35 °C for 5 h, after having reached the late log phase. The specific expression of 0.14 ± 0.02 μg/mL/A600, with a final optical density of 3.43 ± 0.13 A600 (n = 3) was obtained. Purification by nickel affinity chromatography (Hisprep FF) with Imidazole elution followed by size exclusion chromatography (Sephacryl S-100) was carried out connected to an ÄKTA purification system. Quantification was carried out by comparison between the areas under the curve observed in the HPSEC chromatogram, for the unknown samples versus the Internal Standard of rec-hPRL. The final product showed >95% purity by HPSEC analysis. The delta 1-11 G129R-hPRL antagonist was expressed and purified for further in vivo and in vitro tests, in view of clinical applications for inhibiting cancer cell proliferation that overexpresses prolactin receptor and studies related to prolactin function in anterior pituitary.pt_BR
dc.event.siglaBPPpt_BR
dc.format.extent110-110pt_BR
dc.identifier.citationSUZUKI, M.F.; ALMEIDA, L.A.; POMIN, S.A.; SILVA, F.D.; FREIRE, R.P.; OLIVEIRA, J.E.; AFFONSO, R.; BARTOLINI, P.; SOARES, C.R.J. Expression of the human prolactin antagonist delta 1-11 G129R-PRL in E. coli periplasm. In: SANTOS, NATHALIA V. dos (ed.). In: BIOPARTITIONING & PURIFICATION CONFERENCE, November 11-13, 2019, Guarujá, SP. <b>Abstract...</b> São Paulo, SP: Sociedade Brasileira de Microbiologia, 2019. p. 110-110. Disponível em: http://repositorio.ipen.br/handle/123456789/30965.
dc.identifier.orcid0000-0002-6937-1120pt_BR
dc.identifier.orcid0000-0002-7467-3457pt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-9264-4262
dc.identifier.orcidhttps://orcid.org/0000-0002-7982-1789
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/30965
dc.localSão Paulo, SPpt_BR
dc.local.eventoGuarujá, SPpt_BR
dc.publisherSociedade Brasileira de Microbiologiapt_BR
dc.rightsopenAccesspt_BR
dc.titleExpression of the human prolactin antagonist delta 1-11 G129R-PRL in E. coli periplasmpt_BR
dc.typeResumo de eventos científicospt_BR
dspace.entity.typePublication
ipen.autorLARISSA ANDRADE ALMEIDA
ipen.autorFELIPE DOUGLAS DA SILVA
ipen.autorREGINA AFFONSO
ipen.autorCARLOS ROBERTO JORGE SOARES
ipen.autorPAOLO BARTOLINI
ipen.autorJOAO EZEQUIEL DE OLIVEIRA
ipen.autorRENAN PASSOS FREIRE
ipen.autorSTEPHANIE ANGELO POMIN
ipen.autorMIRIAM FUSSAE SUZUKI
ipen.codigoautor14898
ipen.codigoautor14230
ipen.codigoautor1547
ipen.codigoautor509
ipen.codigoautor1503
ipen.codigoautor425
ipen.codigoautor14408
ipen.codigoautor14921
ipen.codigoautor557
ipen.contributor.ipenauthorLARISSA ANDRADE ALMEIDA
ipen.contributor.ipenauthorFELIPE DOUGLAS DA SILVA
ipen.contributor.ipenauthorREGINA AFFONSO
ipen.contributor.ipenauthorCARLOS ROBERTO JORGE SOARES
ipen.contributor.ipenauthorPAOLO BARTOLINI
ipen.contributor.ipenauthorJOAO EZEQUIEL DE OLIVEIRA
ipen.contributor.ipenauthorRENAN PASSOS FREIRE
ipen.contributor.ipenauthorSTEPHANIE ANGELO POMIN
ipen.contributor.ipenauthorMIRIAM FUSSAE SUZUKI
ipen.date.recebimento20-03
ipen.event.datapadronizada2019pt_BR
ipen.identifier.ipendoc26765pt_BR
ipen.notas.internasAbstractpt_BR
ipen.type.genreResumo
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sigepi.autor.atividadeSOARES, C.R.J.:509:810:Npt_BR
sigepi.autor.atividadeBARTOLINI, P.:1503:810:Npt_BR
sigepi.autor.atividadeAFFONSO, R.:1547:820:Npt_BR
sigepi.autor.atividadeOLIVEIRA, J.E.:425:810:Npt_BR
sigepi.autor.atividadeFREIRE, R.P.:14408:810:Npt_BR
sigepi.autor.atividadeSILVA, F.D.:14230:810:Npt_BR
sigepi.autor.atividadePOMIN, S.A.:14921:810:Npt_BR
sigepi.autor.atividadeALMEIDA, L.A.:14898:810:Npt_BR
sigepi.autor.atividadeSUZUKI, M.F.:557:810:Spt_BR
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