JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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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.
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.
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.
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.
First expression of the antagonist of mouse prolactin (S177D-mPRL) by adherent dhfr-CHO cell using p658 vector
2011 - SUZUKI, M.F.; ARTHUSO, F.S.; OLIVEIRA, J.E.; CAPONE, M.V.; DAMIANI, R.; OLIVEIRA, N.A.J.; GOULART RODRIGUES, H.; RIBELA, M.T.C.P.; SOARES, C.R.J.; BARTOLINI, P.
Physico-chemical and biological characterization of glycosylated human prolactin produced at IPEN
2010 - GOULART, H.R.; ARTHUSO, F.S.; CAPONE, M.V.N.; OLIVEIRA, T.L.; OLIVEIRA, J.E.; SUZUKI, M.F.; SOUSA, J.M.; BARTOLINI, P.; SOARES, C.R.J.
A comparison between the oligosaccharide structures of native and recombinant glycosylated human prolactin obtained from CHO cells
2013 - CAPONE, M.V.N.; SUZUKI, M.F.; OLIVEIRA, J.E.; BATOLINI, P.; SOARES, C.R.J.