JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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Human bone morphogenetic protein‑2 (hBMP‑2) characterization by physical–chemical, immunological and biological assays
2020 - SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M. de S.; MAGALHÃES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; SILVA, FABIANA M.; BARTOLINI, PAOLO
Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, sizeexclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived methBMP- 2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.
Human bone morphogenetic protein (hBMP)-2 characterization by physical chemical, immunological and biological assays
2019 - SUZUKI, M.F.; OLIVEIRA, J.E.; DAMIANI, R.; LIMA, E.R.; AMARAL, K.C.; SILVA, F.M.; BARTOLINI, P.
Commercial preparations of human-met-BMP-2 (GenScript) and of CHO-derived hBMP-2 (Infuse-Medtronic) provided a complete characterization of this protein, which belongs to the “transforming growth factors β” superfamily, via SDS-PAGE, Western blotting, reversed-phase HPLC, high-performance size-exclusion chromatography and MALDI-TOF-MS. E.coli-derived met-hBMP-2 has shown a large presence of dimer (MM= 26,054 Da), versus a theoretic value of 26,072 Da. More complex was the distribution of the CHO-derived product, whose exact MM has never been reported due to variable glycosylation: via MALDI-TOF-MS a dimer (28,732 Da) and a large amount of monomer (14,377 Da) were found. A novel method based on RP-HPLC was also validated for hBMP-2 qualitative and quantitative analysis directly in ongoing culture media. The classical “in vitro” bioassay, via alkaline phosphatase induction in murine myoblastic cells C2C12, confirmed that hBMP-2 bioactivity is mostly related to the dimer, being ∼6-fold higher for the CHO-derived glycosylated form. Considering that hBMP-2 is a highly effective osteoinductors, plays an important role during bone regeneration and repair, as well as during embryonic development, and presents an extremely high aggregate value, we believe that these data pave the way to the characterization of this important factor when obtained by DNA recombinant techniques in different host cells.
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.
N-Glycoprofiling analysis for carbohydrate composition and site-occupancy determination in a poly-glycosylated protein: human thyrotropin of different origins
2017 - RIBELA, MARIA T.C.P.; DAMIANI, RENATA; SILVA, FELIPE D.; LIMA, ELIANA R.; OLIVEIRA, JOAO E.; PERONI, CIBELE N.; TORJESEN, PETER A.; SOARES, CARLOS R.; BARTOLINI, PAOLO
Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the -subunit and one in the -subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%–87%) and carbohydrate mass (12%–19%) can be up to 34%–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.
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.
Effects of butyrate and manganese on productivity, sialylation, N-glycosylation site occupancy and biological properties of CHO-derived thyrotropin
2014 - DAMIANI, RENATA; OLIVEIRA, JOAO E.; ALMEIDA, BEATRIZ E.; SANT'ANA, PATRICIA M.; DALMORA, SERGIO L.; BARTOLINI, PAOLO; RIBELA, MARIA T.C.P.
Expression and characterization of mouse prolactin (mPRL) in CHO dhfr-cells
2014 - SUZUKI, MIRIAM F.; SILVA, ALINE B. da; OLIVEIRA, JOAO E. de; ARTHUSO, FERNANDA dos S.; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
Comparative studies of pituitary (NIDDK, USA) and recombinant (Thyrogen and IPEN) human thyroid stimulating hormone (hTSH) for what concerns cabohydrate structures and charge heterogeneity
2006 - OLIVEIRA, J.E.; LOUREIRO, R.F.; CARVALHO, C.M.; DAMIANI, R.; BARTOLINI, P.; RIBELA, M.T.C.P.
A practical and fast adaptation of CHO cells expressing human prolactin to grow in suspension and its application to laboratory production
2011 - ARTHUSO, F.S.; CAPONE, M.V.N.; SUZUKI, M.F.; SOUSA, J.M.; OLIVEIRA, J.E.; BARTOLINI, P.; SOARES, C.R.J.