Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in chinese hamster ovary (CHO) cells

Página do item simplificado
dc.contributor.authorSOARES, C.R.J.pt_BR
dc.contributor.authorGLEZER, A.pt_BR
dc.contributor.authorOKAZAKI, K.pt_BR
dc.contributor.authorUEDA, E.K.M.pt_BR
dc.contributor.authorHELLER, S.R.pt_BR
dc.contributor.authorWALKER, A.M.pt_BR
dc.contributor.authorGOFFIN, V.pt_BR
dc.contributor.authorBARTOLINI, P.pt_BR
dc.coverageInternacionalpt_BR
dc.date.accessioned2014-07-31T11:33:42Zpt_BR
dc.date.accessioned2014-07-31T11:54:27Z
dc.date.available2014-07-31T11:33:42Zpt_BR
dc.date.available2014-07-31T11:54:27Z
dc.date.issued2006pt_BR
dc.description.abstractThe synthesis, puriWcation and characterization of G129R–hPRL and S179D–hPRL, the two better-studied antagonists of human prolactin (hPRL), is described. Both of these have been expressed for the Wrst time, in their authentic form, by a stable CHO cell line, at secretion levels of 7.7 and 4.3 g/106 cells/day, respectively. Previous studies had shown that these hPRL analogs, when produced in bacterial cytoplasm, consistently contained misfolded forms and multimers according to the speciWc denaturation, refolding and puriWcation conditions. These versions also have an N-terminal extra methionine. An extensive physico-chemical characterization was carried out after a practical two-step puriWcation process and included SDS–PAGE and Western blotting analysis, matrix-assisted laser-desorption ionization time-of-Xight mass spectral (MALDI–TOF–MS) analysis, high-performance size-exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography (RP–HPLC). This last technique revealed a considerable diVerence in hydrophobicity due to a single amino acid substitution, with S179D–hPRL less (tRR D 0.85 § 0.010) and G129R–hPRL more (tRR D 1.10§ 0.013) hydrophobic than hPRL, where tRR is the relative retention time. The biological characterization was based on further reWnement of a sensitive proliferation assay using the pro-B murine cell line (Ba/F3) transfected with the long form hPRL receptor cDNA such that the minimal detectable dose was 0.04 ng of hPRL/mL, the Ba/F3-LLP assay. On the basis of this assay, the relative residual agonistic activity of these two products, determined against a hPRL international standard in four independent assays, was 53 £ 10¡3 for S179D–hPRL and 70£ 10¡5 for G129R–hPRL. We believe that the present synthesis and characterization could be extremely helpful for studies of these two proteins, which have been reported to antagonize tumor growth-promoting eVects of hPRL in vivo in animal models of breast and prostate cancer.
dc.format.extent182-194pt_BR
dc.identifier.citationSOARES, C.R.J.; GLEZER, A.; OKAZAKI, K.; UEDA, E.K.M.; HELLER, S.R.; WALKER, A.M.; GOFFIN, V.; BARTOLINI, P. Physico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in chinese hamster ovary (CHO) cells. <b>Protein Expression and Purification</b>, v. 48, n. 2, p. 182-194, 2006. DOI: <a href="https://dx.doi.org/10.1016/j.pep.2006.04.013">10.1016/j.pep.2006.04.013</a>. Disponível em: http://repositorio.ipen.br/handle/123456789/7693.
dc.identifier.doi10.1016/j.pep.2006.04.013
dc.identifier.fasciculo2pt_BR
dc.identifier.issn1046-5928pt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-7982-1789
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/7693pt_BR
dc.identifier.vol48pt_BR
dc.relation.ispartofProtein Expression and Purificationpt_BR
dc.rightsopenAccessen
dc.subjectlthpt_BR
dc.subjecthormonespt_BR
dc.subjectcho cellspt_BR
dc.subjectmammary glandspt_BR
dc.subjectprostaglandinspt_BR
dc.subjectneoplasmspt_BR
dc.titlePhysico-chemical and biological characterizations of two human prolactin analogs exhibiting controversial bioactivity, synthesized in chinese hamster ovary (CHO) cellspt_BR
dc.typeArtigo de periódicopt_BR
dspace.entity.typePublication
ipen.autorPAOLO BARTOLINI
ipen.autorSUSANA DA ROCHA HELLER
ipen.autorERIC KINNOSUKE MARTINS UEDA
ipen.autorCARLOS ROBERTO JORGE SOARES
ipen.autorKAYO OKAZAKI
ipen.codigoautor1503
ipen.codigoautor3484
ipen.codigoautor2072
ipen.codigoautor509
ipen.codigoautor1220
ipen.contributor.ipenauthorPAOLO BARTOLINI
ipen.contributor.ipenauthorSUSANA DA ROCHA HELLER
ipen.contributor.ipenauthorERIC KINNOSUKE MARTINS UEDA
ipen.contributor.ipenauthorCARLOS ROBERTO JORGE SOARES
ipen.contributor.ipenauthorKAYO OKAZAKI
ipen.date.recebimento06-09pt_BR
ipen.identifier.fi1.867pt_BR
ipen.identifier.ipendoc11417pt_BR
ipen.identifier.iwosWoSpt_BR
ipen.range.fi1.500 - 2.999
ipen.type.genreArtigo
relation.isAuthorOfPublication7d228133-8477-43fb-941d-2cfb6a48c46c
relation.isAuthorOfPublication5c1adbd6-5604-4ead-b9ab-7aaa3e9c27a7
relation.isAuthorOfPublication1510e048-7519-4f39-9fc1-dff221c4124a
relation.isAuthorOfPublicationd6719ab2-f2e9-4d7c-9cea-a6316fc14c9e
relation.isAuthorOfPublication131eab71-499a-48c8-8f16-46ffafd0608f
relation.isAuthorOfPublication.latestForDiscoveryd6719ab2-f2e9-4d7c-9cea-a6316fc14c9e
sigepi.autor.atividadeSOARES, C.R.J.:509:18:Spt_BR
sigepi.autor.atividadeOKAZAKI, K.:1220:18:Npt_BR
sigepi.autor.atividadeUEDA, E.K.M.:2072:18:Npt_BR
sigepi.autor.atividadeHELLER, S.R.:3484:18:Npt_BR
sigepi.autor.atividadeBARTOLINI, P.:1503:18:Npt_BR
Pacote Original
Agora exibindo 1 - 1 de 1
Imagem de Miniatura
Nome:
11417.pdf
Tamanho:
449.52 KB
Formato:
Adobe Portable Document Format
Baixar
Coleções
Periódicos