CARLOS ROBERTO JORGE SOARES

CARLOS ROBERTO JORGE SOARES

(Fonte: Lattes)

Resumo

Graduado em Farmácia e Bioquímica pela Universidade de São Paulo (1989), realizou Mestrado (1995) e Doutorado (2000) em Tecnologia Nuclear - Aplicações pela Universidade de São Paulo. Atualmente é pesquisador do Instituto de Pesquisas Energéticas (IPEN-CNEN/SP) e professor de pós-graduação vinculado à Universidade de São Paulo. Com experiência em biotecnologia na expressão de proteínas recombinantes por bactéria e por células de mamífero. Atua principalmente no seguinte tema: síntese, purificação, caracterização e aplicações de hormônios recombinantes. Atualmente é Gerente do Centro de Biotecnologia do IPEN. (Texto extraído do Currículo Lattes em 08 out. 2021)

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Periplasmic synthesis and purification of the human prolactin antagonist Δ1‑11‑G129R‑hPRL
2021 - SUZUKI, MIRIAM F.; ALMEIDA, LARISSA A.; POMIN, STEPHANIE A.; SILVA, FELIPE D.; FREIRE, RENAN P.; OLIVEIRA, JOAO E.; AFFONSO, REGINA; SOARES, CARLOS R.J.; BARTOLINI, PAOLO
The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.
Purificação de antagonista de prolactina expresso em bactérias Escherichia coli
2019 - ALMEIDA, LARISSA A.; SOARES, CARLOS R.J.
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.
High production and optimization of the method for obtaining pure recombinant human prolactin
2018 - AFFONSO, REGINA; SOARES, CARLOS R.; RIBELA, MARIA T.; BARTOLINI, PAOLO
Prolactin is a pituitary hormone that is involved diverse physiological functions, such as lactation, reproduction, metabolism, osmoregulation, immunoregulation, and behavior. Its level of glycosylation is low in vivo, which favors its expression in bacterial systems. In the present work recombinant human prolactin (rec-hPRL) was expressed from the p1813-hPRL vector in Escherichia coli strain in inclusion bodies with 530.67 mg of rec-hPRL per liter of induced bacterial culture. The solubilization and renaturation of rec-hPRL followed by two methods described in the literature for this protein: one with detergent and basic pH, and other urea and dialyses was done by studying. The protocol with detergent/basic pH was not successful, whereas protocol with urea/dialyses was obtained pure protein and this was optimized. Rec-hPRL was obtained in a soluble, pure and active form, when the sample was 8-fold concentrated in the solubilization phase, allowing 33% recovery, 3-fold more that the original method. The pure protein was obtained with 38.37 i. u./mg activity, which is three times greater than that of the PRL standard from the WHO. In conclusion, this work obtained the highest production of rechPRL, and concentrating the sample eight times in the solubilization stage was decisive for obtaining a highly concentrated, active protein for future work.
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.
Distribution of growth hormone-responsive cells in the mouse brain
2017 - FURIGO, ISADORA C.; METZGER, MARTIN; TEIXEIRA, PRYSCILA D.S.; SOARES, CARLOS R.J.; DONATO JUNIOR, JOSE
Growth hormone (GH) exerts important biological effects primarily related to growth and metabolism. However, the role of GH signaling in the brain is still elusive. To better understand GH functions in the brain, we mapped the distribution of GH-responsive cells and identified the receptors involved in GH central effects. For this purpose, mice received an acute intraperitoneal challenge with specific ligands of the GH receptor (mouse GH), prolactin receptor (prolactin) or both receptors (human GH), and their brains were subsequently processed immunohistochemically to detect the phosphorylated form of STAT5 (pSTAT5). GH induced pSTAT5 immunoreactivity in neurons, but not in astroglial cells of numerous brain regions, including the cerebral cortex, nucleus accumbens, hippocampus, septum and amygdala. The most prominent populations of GH-responsive neurons were located in hypothalamic areas, including several preoptic divisions, and the supraoptic, paraventricular, suprachiasmatic, periventricular, arcuate, ventromedial, dorsomedial, tuberal, posterior and ventral premammillary nuclei. Interestingly, many brainstem structures also exhibited GH-responsive cells. Experiments combining immunohistochemistry for pSTAT5 and in situ hybridization for GH and prolactin receptors revealed that human GH induced pSTAT5 in most, but not all, brain regions through both prolactin and GH receptors. Additionally, males and females exhibited a similar number of GH-responsive cells in forebrain structures known to be sexually dimorphic. In summary, we found GH-responsive cells primarily distributed in brain regions implicated in neurovegetative, emotional/motivational and cognitive functions. Our findings deepen the understanding of GH signaling in the brain and suggest that central GH signaling is likely more ample and complex than formerly recognized
Uncovering the mechanism of action of the anti-diabetic effects of bromocriptine: the role of prolactin
2014 - FURIGO, ISADORA C.; DONATO, JOSE; SUZUKI, MIRIAM F.; ZAMPIERI, THAIS T.; PEDROSO, JOAO A.B.; LOBO, ANGELA M.R.; ALENCAR, AMANDA; OLIVEIRA, JOAO E.; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
N-glycoprofiling analysis in a simple glycoprotein model: glycosylated human prolactin
2014 - CAPONE, MARCOS V.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; SOARES, CARLOS R.; BARTOLINI, PAOLO
Aplicação dos bioensaios com células Ba/F3-LLP e Nb2 na determinação da potência biológica da prolactina humana
2008 - ARTHUSO, FERNANDA dos S.; SOARES, CARLOS R.J.
Aplicação do bioensaio Ba/F3-LLP na determinação da potência biológica da prolactina humana glicosiladfa (G-hPRL)
2007 - ARTHUSO, FERNANDA dos S.; SOARES, CARLOS R.J.