Point mutation of serine 179 in the human Prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacities

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dc.contributor.authorUEDA, ERICpt_BR
dc.contributor.authorSOARES, CARLOSpt_BR
dc.contributor.authorWALKER, AMEAEpt_BR
dc.contributor.authorBARTOLINI, PAOLOpt_BR
dc.coverageInternacionalpt_BR
dc.date.accessioned2014-07-15T13:48:07Zpt_BR
dc.date.accessioned2014-07-30T11:51:31Z
dc.date.available2014-07-15T13:48:07Zpt_BR
dc.date.available2014-07-30T11:51:31Z
dc.date.issued2006pt_BR
dc.description.abstractBackground: S179D prolactin (S179D PRL) is a pseudophosphorylated form of human prolactin (PRL) which has inhibitory effects on tumor growth [1] and angiogenesis [2]. The S179D PRL preparations used for these experiments consisted of properly refolded inclusion bodies (IB) from Escherichia coli [3]. Trying to attain a better folded mutant, we used secretion expression based systems. However, single point mutations can affect protein periplasmic expression [4], and secretion from mammalian cells [5]. We observed that upon a mutation of Serine 179 to an Aspartate, expression was nearly abolished when compared with PRL in E. coli periplasm, while the cytoplasmic product was more prone to proteolysis. Using eukaryotic cells we were able to produce preparations comparable to IBs in terms of bioactivity. We also demonstrated that this mutant had a higher affinity for heparin and lower binding capacity towards divalent metals (M (II)). Results: S179D PRL periplasmic expression was very low when compared to PRL. Use of different promoters, different signal peptides or different activation temperatures had no effect (figure 1). MALDI-TOF spectrometry was carried out for identity of S179D PRL in the extracts (figure 2). BL21 strain was used (figure 1B) without improvements for S179D PRL expression (table 2). We used BL 21 codon plus® in order to investigate the GC-, AT-rich sequence of the PRLs influence on expression. This strain did not rescue expression of S179D PRL or PRL (figure 1C). pTac induction at lower temperatures should encourage protein solubility and folding in the cytoplasm [6]. We carried out cytoplasmic expression with an Origami B strain, in which cytoplasm folding is facilitated [7]. Surprisingly, when S179D PRL was produced in soluble form, unlike PRL, low molecular forms were observed (figure 3A and 3B), and also in BL21, cleaved forms and soluble high molecular aggregates were present (figure 3A). pL constructs had very low yields for both PRLs (figure 3). An eukaryotic expression system was chosen to successfully produce soluble, monomeric, recombinant S179D PRL. B-casein bioassays were carried out to check S179D PRL folding. (figure 5). Moreover S179D PRL had a decreased affinity towards Ni (II) ans Zn (II). On the other hand it had an increased affinity towards heparin. Conclusion: We tried to produce a correctly folded form of S179D PRL, already obtained as refolded IBs [3]. Unexpectedly, this point mutation of PRL impaired protein expression, and was not related to the strain, protease degradation of our protein, or preferential codon usage (figure 1). To avoid proteolysis and misfolding we used lower temperatures during protein production [8], but it failed to produce S179D PRL. Low levels of S179D PRL were only detected by immunoblots (figure 1) and by immunoassay (table 1). Expression of soluble S179D PRL in the cytoplasm of E.coli was not efficient either, as denoted by soluble aggregates and cleaved S179D PRLs. Eukaryotic expression systems have a better folding machinery, being difficult-to-fold proteins more easily expressed [9]. Thus, we successfully produced S179D PRL at RP-HPLC detectable levels (figure 4). MALDI-TOF analysis showed that all samples had the expected molecular weight (figure 2). RP-HPLC demonstrated that S179D PRL had a different folding than PRL. The bioactivity assay showed that all preparations of S179D PRL were correctly folded. S179D PRL also showed physical-chemical differences, having a lower M (II)-affinity and a higher heparin-affinity. This confirms reports of PRL mutants with low Zn (II) affinity that are poorly secreted [4] and also could account for its anti-angiogenic effect [2, 10].
dc.format.extent50-54pt_BR
dc.identifier.citationUEDA, ERIC; SOARES, CARLOS; WALKER, AMEAE; BARTOLINI, PAOLO. Point mutation of serine 179 in the human Prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacities. <b>Microbial Cell Factories</b>, v. 5, p. 50-54, 2006. Suppl. 1. DOI: <a href="https://dx.doi.org/10.1186/1475-2859-5-S1-P17">10.1186/1475-2859-5-S1-P17</a>. Disponível em: http://repositorio.ipen.br/handle/123456789/5438.
dc.identifier.doi10.1186/1475-2859-5-S1-P17
dc.identifier.issn1475-2859pt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-7982-1789
dc.identifier.suplementoSuppl. 1pt_BR
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/5438pt_BR
dc.identifier.vol5pt_BR
dc.relation.ispartofMicrobial Cell Factoriespt_BR
dc.rightsopenAccessen
dc.subjectgene mutationspt_BR
dc.subjectserinept_BR
dc.subjectlthpt_BR
dc.subjectproteinspt_BR
dc.subjectrecombinant dnapt_BR
dc.subjectescherichia colipt_BR
dc.subjectproteolysispt_BR
dc.subjectheparinpt_BR
dc.subjectimmunoassaypt_BR
dc.titlePoint mutation of serine 179 in the human Prolactin (PRL) affects recombinant protein expression, folding and secretion, abolishes PRL nickel (II)-binding and increases heparin binding capacitiespt_BR
dc.typeArtigo de periódicopt_BR
dspace.entity.typePublication
ipen.autorPAOLO BARTOLINI
ipen.autorCARLOS ROBERTO JORGE SOARES
ipen.autorERIC KINNOSUKE MARTINS UEDA
ipen.codigoautor1503
ipen.codigoautor509
ipen.codigoautor2072
ipen.contributor.ipenauthorPAOLO BARTOLINI
ipen.contributor.ipenauthorCARLOS ROBERTO JORGE SOARES
ipen.contributor.ipenauthorERIC KINNOSUKE MARTINS UEDA
ipen.date.recebimento06-11pt_BR
ipen.identifier.fiSem F.I.pt_BR
ipen.identifier.ipendoc11490pt_BR
ipen.type.genreArtigo
relation.isAuthorOfPublication7d228133-8477-43fb-941d-2cfb6a48c46c
relation.isAuthorOfPublicationd6719ab2-f2e9-4d7c-9cea-a6316fc14c9e
relation.isAuthorOfPublication1510e048-7519-4f39-9fc1-dff221c4124a
relation.isAuthorOfPublication.latestForDiscovery1510e048-7519-4f39-9fc1-dff221c4124a
sigepi.autor.atividadeUEDA, ERIC:2072:18:Spt_BR
sigepi.autor.atividadeSOARES, CARLOS:509:18:Npt_BR
sigepi.autor.atividadeBARTOLINI, PAOLO:1503:18:Npt_BR
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