JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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Expression of glycosylated human prolactin in HEK293 cells and related N‑glycan composition analysis
2019 - SILVA, FELIPE D.; OLIVEIRA, JOÃO E.; FREIRE, RENAN P.; SUZUKI, MIRIAM F.; SOARES, CARLOS R.; BARTOLINI, PAOLO
Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO® vector carrying the hPRL cDNA. A concentration of ~ 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with ~ 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL (~ 10%) and falling within the wide range of 5–30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI–TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was ~ sevenfold lower, but slightly higher than that observed in native G-hPRL. The “in vitro” bioactivity of HEK-G-hPRL was ~ fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures.
Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination
2018 - SANTANA, P.M.; OLIVEIRA, J.E.; LIMA, E.R.; SOARES, C.R.J.; PERONI, C.N.; BARTOLINI, P.; RIBELA, MARIA T.C.P.
A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and β-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.
Transient expression of recombinant human prolactin and thyrotropin in human embryonic kidney (Expi293FTM) suspension cells
2017 - SILVA, F.D.; SEVILHANO, T.C.A.; FREIRE, R.P.; SUZUKI, M.F.; OLIVEIRA, J.E.; PERONI, C.N.; RIBELA, M.T.C.P.; BARTOLINI, P.; SOARES, C.R.J.
Human prolactin (hPRL) and human thyrotropin (hTSH) are pituitary polypeptide hormones with key functions in the physiological regulation of the human body. hPRL is highly secreted during lactation, has important action in reproduction and for immunoregulation, among other functions. hTSH is related to the control of thyroid gland. The Chinese Hamster Ovary (CHO) and Human Embryonic Kidney (HEK293) cells are the most used hosts for expression of recombinant human proteins because they can be easily cultured in suspension conditions, and express high levels of proteins that have a relative similarity in post-translational modifications compared to their human counterparts. Our laboratory has experience in the synthesis of these proteins in the Escherichia coli periplasm (hPRL), adhered CHO (hPRL and hTSH), suspension CHO (hPRL) and adhered HEK293T cells (hTSH). The aim of this work was to produce hPRL and hTSH in suspension Expi293FTM cells for their characterization. The hPRL and hTSH cDNA were introduced into the commercial plasmid pcDNATM 3.4-TOPO® and 30 μg of these plasmids were used to transfect 30 mL of suspension Expi293FTM cells (2.5 x 106 cells/mL) in a 125 mL erlenmeyer, using 81 μL of ExpiFectamineTM transfection agent. After 16 h of transfection, 150 μL of Enhancer 1 and 1.5 mL of Enhancer 2 were added and the culture was maintained in an incubator at 37 °C, 8% CO2, at 125 rpm in orbital shaker. Samples of conditioned media (Expi293TM expression medium) were collected during 4 days and stored at -80 °C. These were analyzed by SDS-PAGE, ELISA, Western blotting, and HPLC. For the first time, hPRL and hTSH, were transiently expressed in human (Expi293FTM) suspension cells, the expression levels reaching, on the 3rd day, 46 μg of hPRL/mL and 116 μg of hTSH/mL. These results show that the expression is clearly dependent on the characteristics of the protein and that this methodology is very efficient to obtain high levels of human glycoproteins in a short time and will allow us to purify them and compare their glycosylation profiles of these to CHO-derived and human native pituitary hormones.
Molecular cloning and characterization of pirarucu (Arapaima gigas) follicle-stimulating hormone and luteinizing hormone β-subunit cDNAs
2017 - SEVILHANO, THAIS; CARVALHO, ROBERTO F. de; OLIVEIRA, NELIO A. de J.; OLIVEIRA, JOAO E.; MALTAROLLO, VINICIUS G.; TROSSINI, GUSTAVO; GARCEZ, RIVIANE; BARTOLINI, PAOLO
The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called ªseat-beltº, favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.
Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space
2017 - MENEZES, ANA C.S.C.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; RIBELA, MARIA T.C.P.; FURIGO, ISADORA C.; DONATO JUNIOR, JOSE; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
The human growth hormone receptor antagonist G120R-hGH precludes dimerization of GH and prolactin receptors and consequently JAK/STAT signaling. Some modifications in this antagonist resulted in a drug specific for the GH receptor, called Pegvisomant (Somavert®). However, the original G120R-hGH is usually synthesized in bacterial cytoplasm as inclusion bodies, not being a commercial product. The present work describes the synthesis and characterization of G120R-hGH secreted into bacterial periplasm and obtained with a vector based on a constitutive lambda-PL promoter. This antagonist can be useful for studies aiming at investigating the effects of a simultaneous inhibition of GH and prolactin signaling, as a potential anti-tumoral or anti-diabetic compound. G120R-hGH, synthesized using the W3110 E. coli strain, showed a yield of 1.34 ± 0.24 mg/ml/A600 (~0.79 mg G120R-hGH/g of wet weight cells) after cultivation at 30 C up to 3 A600 units and induction at 37 C, for 6 h, with final 4.3 ± 0.3 A600. A laboratory scale purification was carried out using three chromatographic steps with a total yield of 32%, reaching 98% purity. The obtained protein was characterized by SDS-PAGE, Western Blotting, Mass spectrometry, RP-HPLC, HPSEC and in vitro proliferation bioassay. The proliferation assay, based on Ba/F3- LLP cells, shows that G120R-hGH (100 ng/ml) significantly inhibited (64%) the proliferative action of hGH (1 ng/ml). This is the first time that G120R-hGH is synthesized in bacterial periplasmic space and therefore correctly folded, without the initial methionine. The reasons for a divergent efficacy for antagonizing hGH versus hPRL is currently unknown and deserves further investigation.
Expressão do antagonista de receptor do hormônio do crescimento humano G120R-hGH no espaço periplásmico de Escherichia coli e sua caracterização físico-química
2015 - MENEZES, ANA C.S.C.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; RIBELA, MARIA T.C.P.; FURIGO, ISADORA C.; DONATO JUNIOR, JOSE; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
N-glycoprofiling analysis in a single glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin
2015 - CAPONE, MARCOS V.N.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; SOARES, CARLOS R.J.; BARTOLINI, PAOLO
N-glycoprofiting analysis in a single ghycoprotein model: Glycosylated human prolactin
2014 - CAPONE, MARCOS V.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; SOARES, CARLOS R.; BARTOLINI, PAOLO
The influence of Escherichia coli cultivation temperature on interferon alpha 2a expression (IFN-'alfa'2a)
2014 - ARTHUSO, FERNANDA dos S.; SUZUKI, MIRIAM F.; OLIVEIRA, NELIO A. de J.; OLIVEIRA, JOAO E. de; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
Purification and characterization recombinant glycosylated human prolactin synthesized in cho cells
2008 - OLIVEIRA, T.L.; HELLER, S.R.; OLIVEIRA, J.E.; ARTHUSO, F.S.; GOULART, H.R.; SOUSA, J.M.; SUZUKI, M.F.; BARTOLINI, P.; SOARES, C.R.S.