ERIC KINNOSUKE MARTINS UEDA
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Resumo IPEN-doc 11502 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 capacities2006 - UEDA, ERIC; SOARES, CARLOS; WALKER, AMEAE; BARTOLINI, P.Resumo IPEN-doc 11011 Reversed-phase high performance liquid chromatography (RP-HPLC) analysis and hydrophobicity studies of recombinant human pituitary hormones synthesized in E. coli and CHO cells2005 - RIBELA, M.T.C.P.; CARVALHO, C.M.; HELLER, S.R.; LOUREIRO, R.F.; OLIVEIRA, J.E.; OZAKI, N.A.; PERONI, C.N.; SOARES, C.R.J.; SOUZA, J.M.; UEDA, E.K.; BARTOLINI, P.The synthesis and laboratory production of human growth hormone (hGH) and prolactin (hPRL) have been carried out in genetically modified E. coli, while those of thyroid stimulating hormone (hTSH) and of two analogs antagonists of hPRL (G129RhPRL and S179D-hPRL) in stably transfected CHO cells. Human follicle stimulating hormone (hFSH) and luteinizing hormone (hLH) have not been synthesized yet in our laboratory but their HPLC analytical methodologies are under development. For the purpose of studying and improving synthesis and bioreaction yields and, at the same time, planning and following all subsequent purification steps, novel RP-HPLC methods have been set up for each hormone. For hGH and hPRL, isocratic RP-HPLC methods have been developed that can qualitatively and quantitatively analyze the two hormones directly in osmotic shock fluids, already during fermentation. For hTSH, hFSH and hLH, RP-HPLC gradient elutions have been set for the analysis of these hormones in their purified form and in CHO conditioned medium. For these three glycoproteins hydrophobicities have been compared and the following order established: hLH>hTSH>hFSH. An analogous hydrophobicity index has been also determined for hPRL and its analogs, being G129R-hPRL>hPRL>S179D-hPRL. Still concerning hFSH, for the first time it has been possible to optimize RP-HPLC elution conditions that are able to preserve its undissociated heterodimeric structure. Thanks to this tool it was thus possible to carry out a comparative study on pituitary, urinary and CHO-derived hFSH preparations, revealing differences that are probably due to the carbohydrate moiety, as already observed for hTSH. Classical highperformance size exclusion chromatography (HPSEC) together with MALDI-TOF-MS analysis was also employed along with these studies, to complement physico-chemical characterization of our proteins of interest.Artigo IPEN-doc 11490 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 capacities2006 - UEDA, ERIC; SOARES, CARLOS; WALKER, AMEAE; BARTOLINI, PAOLOBackground: 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].