JOAO EZEQUIEL DE OLIVEIRA

JOAO EZEQUIEL DE OLIVEIRA

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Isolation and characterization of the Arapaima gigas growth hormone (ag-GH) cDNA and three-dimensional modeling of this hormone in comparison with the human hormone (hGH)
2023 - LIMA, ELIANA R.; FREIRE, RENAN P.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; YOSIDAKI, VANESSA L.; PERONI, CIBELE N.; SEVILHANO, THAIS; ZORZETO, MOISES; TORATI, LUCAS S.; SOARES, CARLOS R.J.; LIMA, IGOR D. de M.; KRONENBERGER, THALES; MALTAROLLO, VINICIUS G.; BARTOLINI, PAOLO
In a previous work, the common gonadotrophic hormone α-subunit (ag-GTHα), the ag-FSH β- and ag-LH β-subunit cDNAs, were isolated and characterized by our research group from A. gigas pituitaries, while a preliminary synthesis of ag-FSH was also carried out in human embryonic kidney 293 (HEK293) cells. In the present work, the cDNA sequence encoding the ag-growth hormone (ag-GH) has also been isolated from the same giant Arapaimidae Amazonian fish. The ag-GH consists of 208 amino acids with a putative 23 amino acid signal peptide and a 185 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the Elopiformes (82.0%), followed by Anguilliformes (79.7%) and Acipenseriformes (74.5%). The identity with the corresponding human GH (hGH) amino acid sequence is remarkable (44.8%), and the two disulfide bonds present in both sequences were perfectly conserved. Three-dimensional (3D) models of ag-GH, in comparison with hGH, were generated using the threading modeling method followed by molecular dynamics. Our simulations suggest that the two proteins have similar structural properties without major conformational changes under the simulated conditions, even though they are separated from each other by a >100 Myr evolutionary period (1 Myr = 1 million years). The sequence found will be used for the biotechnological synthesis of ag-GH while the ag-GH cDNA obtained will be utilized for preliminary Gene Therapy studies.
Molecular cloning and AlphaFold modeling of thyrotropin (ag-TSH) from the Amazonian fish Pirarucu (Arapaima gigas)
2023 - FREIRE, RENAN P.; HERNANDEZ-GONZALEZ, JORGE E.; LIMA, ELIANA R.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E. de; TORATI, LUCAS S.; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TSH) is a glycoprotein hormone formed by 2 subunits α and β whose main activity is related to the synthesis of thyroid hormones (THs)—T3 and T4. In this work, we present a combination of bioinformatics tools to identify Arapaima gigas βTSH (ag-βTSH), modeling its molecular structure and express the recombinant heterodimer form in mammalian cells. Using the combination of computational biology, based on genome-related information, in silico molecular cloning and modeling led to confirm results of the ag-βTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-βTSH retrieved 146 amino acids with a signal peptide of 21 amino acid residues and 6 disulfide bonds. The sequence has a similarity to 39 fish species, ranging between 43.1% and 81.6%, whose domains are extremely conserved, such as cystine knot motif and N-glycosylation site. The Arapaima gigas thyrotropin (ag-TSH) model, solved by AlphaFold, was used in molecular dynamics simulations with Scleropages formosus receptor, providing similar values of free energy ΔGbind and ΔGPMF in comparison with Homo sapiens model. The recombinant expression in HEK293F cells reached a yield of 25 mg/L, characterized via chromatographic and physical-chemical techniques. This work shows that other Arapaima gigas proteins could be studied in a similar way, using the combination of these techniques, recovering more information from its genome and improving the reproduction and preservation of this prehistoric fish.
High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization
2022 - CHURA-CHAMBI, ROSA M.; PRIETO-DA-SILVA, ALVARO R. de B.; DI LELA, MATHEUS M.; OLIVEIRA, JOAO E.; ABREU, PATRICIA E.A.; MEIRELES, LUCIANA R.; ANDRADE JUNIOR, HEITOR F. de; MORGANTI, LIGIA
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293
2021 - FREIRE, RENAN P.; SILVA, FELIPE D.; VITALE, PHELIPE; TODESCHINI, IRIS; MENEZES, FILIPE; OLIVEIRA, JOAO E. de; OLIVEIRA, MONA das N.; SOARES, CARLOS R.J.
SARS-CoV-2 is a zoonotic virus RNA positive, which became responsible to be the largest sanitary crisis faced by humanity: Coronavirus disease 2019 (COVID-19). Some symptoms include major sneeze conditions, who could evolve to severe acute respiratory syndrome, and in some cases, to death. Techniques for accurate detection of this virus are essential to promote an accurate diagnosis of infected patients. SARS-CoV-2 has several targets with clinical interest; although, the focuses is on Spike (S), a homotrimer glycoprotein, that interacts with angiotensin converting enzyme receptor (ACE2), developing the infection in host cells. Thus, we recognize that the demand for the glycoprotein S is necessary, requiring large amounts with high purity level. The current work has the main objective the transient expression of SARS-CoV-2 S protein into suspension human embryonic kidney cells 293 (HEK293), purification and characterization, to use it as a template for discovering new molecular markers. SARSCoV- 2 S modified protein cDNA was inserted into pαH plasmid, amplified, and purified. For transient recombinant protein expression, 7.5 x 107 HEK293 cells (Expi293FTM cells) was seeded in 27 mL Expi293™ culture Medium. The transfection was carried out with a cationic lipid ExpiFectamine™ and 30 μg of plasmid, mixed with 3 mL Opti-Mem® culture medium. Cell culture was maintained for seven days in 125 mL vent cap Erlenmeyer, 32 ºC, 8% CO2, under 125 rpm orbital shaker rotation. 10 mL aliquots were collected on four- and seven-day post transfection, stored at -80 ºC. Physical-chemical and biological characterizations were determined by SDS-PAGE, ELISA, and Western Blotting. Purification from 40 mL of conditioned medium was carried out in two steps: Strep-Tag affinity column, followed by a size exclusion Superose® 6 (10/300), 5.0 mg of oligomeric recombinant protein with 95% purity was obtained. We believe that this process can be easily adapted to different volumes, being very useful for obtaining, in a short time, enough pure and immunological active SARS-CoV-2 S for further studies and applications, such as, cryogenic electron microscopy, mass spectroscopy, N-glycan structures, antibody production and immunologic assays development.
Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space
2021 - OLIVEIRA, JOAO E.; SUZUKI, MIRIAM F.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M.S.; MAGALHAES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; BARTOLINI, PAOLO
Human BMP-2, a homodimeric protein that belongs to the TGF- family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.
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.
Variations in essential elements after malignant transformation of kidney epithelial tubular cells
2020 - BELLINI, MARIA H.; SOUZA, ALEXANDRE L. de; SILVA, FABIO F. da; GUILHEN, SABINE N.; FERREIRA, RAFAEL V. de P.; ARAUJO, LEANDRO G. de; OLIVEIRA, JOAO E. de; MARUMO, JULIO T.
Cell line-based research is a valuable tool for the study of cancer physiopathology and the discovery of new drugs for use in clinical practice. In this study, inductively coupled plasma mass spectrometry (ICP-MS) was used to estimate Ca, Co, Cu, Fe, K, Mg, Mn, Na, P, S, Se, and Zn in epithelial tubular cells (HK-2) and kidney tumor cells (Caki-1 cells). The most relevant difference was a decrease in the contents of Ca, Cu, Fe, K, Mg, Mn, Na, P, S, and Zn. A significant accumulation of Co was also detected in Caki-1 cells. The fold change variation of each element concentration between HK-2 and Caki-1 cells was Ca (‒0.40), Co (1.37), Cu (‒0.68), Fe (‒0.56), K (‒0.40), Mg (‒0.41), Mn (-0.54), Na (‒0.33), P (‒0.31), S (‒0.26), and Zn (‒0.73). These findings indicate that the elements mainly affect the metabolic pathways of epithelial kidney cells. Thus, our findings open a new avenue for RCC target therapy
Essential elements as biomarkers of metastatic renal cell carcinoma
2020 - BELLINI, MARIA H.; SOUZA, ALEXANDRE L. de; SILVA, FABIO F. da; GUILHEN, SABINE N.; FERREIRA, RAFAEL V. de P.; OLIVEIRA, JOAO E. de; MARUMO, JULIO T.
Renal cell carcinoma (RCC) represents 3% of human malignant tumors and approximately 90% of malignant renal neoplasms. Despite great therapeutic advances in the last decade, metastatic RCC (mRCC) is still considered an incurable disease. In this study, we examined the potential of essential elements as biomarkers of mRCC using an orthotropic metastatic mouse model. Frozen lung and plasma samples from healthy and mRCC-induced mice were lyophilized, digested, and analyzed using inductively coupled plasma mass spectrometry. In metastatic lungs, a significant increase in Ca concentration (268%) was observed, whereas a significant decrease in Cu (23.2%), Fe (17.4%), Mn (38.8%), and Na (11.7%) was observed. The plasma of mRCC-induced mice showed decreased concentrations of Mn (53%), Na (19.7%) and Zn (49,50%) and increased levels of Ca (53%), Cu (39.5%), Our findings revealed marked differences in the concentrations of essential elements in the lung and plasma of the metastatic mouse model. The circulating levels of Ca, Cu, Mn, Na, and Zn could be utilized as diagnostic and therapeutic response biomarkers.
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.
Expression of the human prolactin antagonist delta 1-11 G129R-PRL in E. coli periplasm
2019 - SUZUKI, M.F.; ALMEIDA, L.A.; POMIN, S.A.; SILVA, F.D.; FREIRE, R.P.; OLIVEIRA, J.E.; AFFONSO, R.; BARTOLINI, P.; SOARES, C.R.J.
Recombinant human prolactin antagonist delta 1-11 G129R-hPRL is a 21.9 kDa 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 an authentic, soluble, and correctly folded protein, as an alternative to the cytoplasmic production in inclusion bodies of an unfolded, insoluble protein, carrying an extra initial methionine. The aim of this work was to carry out the expression of delta 1-11 G129R-hPRL antagonist in the periplasm of E. coli, testing different temperatures. E. coli BL21(DE) strain, transformed with a plasmid based on a pET25b(+) vector, DsbA signal peptide and delta 1-11 G129R-hPRL cDNA, was cultured in LB medium with ampicillin addition. After overnight culture at 30 °C, 0.6 mM IPTG was added and five different temperatures were applied: 25, 30, 32, 35 and 37 °C. Periplasmic fluid was extracted after 5 hours by osmotic shock. The samples were analyzed by SDS-PAGE, Western blotting and RP-HPLC. The best condition was increasing the temperature to 35 °C for 5 h, after having reached the late log phase. The specific expression of 0.14 ± 0.02 μg/mL/A600, with a final optical density of 3.43 ± 0.13 A600 (n = 3) was obtained. Purification by nickel affinity chromatography (Hisprep FF) with Imidazole elution followed by size exclusion chromatography (Sephacryl S-100) was carried out connected to an ÄKTA purification system. Quantification was carried out by comparison between the areas under the curve observed in the HPSEC chromatogram, for the unknown samples versus the Internal Standard of rec-hPRL. The final product showed >95% purity by HPSEC analysis. The delta 1-11 G129R-hPRL antagonist was expressed and purified for further in vivo and in vitro tests, in view of clinical applications for inhibiting cancer cell proliferation that overexpresses prolactin receptor and studies related to prolactin function in anterior pituitary.