STEPHANIE ANGELO POMIN

STEPHANIE ANGELO POMIN

Página do item completo

Resultados de Busca

Agora exibindo 1 - 5 de 5

Síntese de prolactina humana em citoplasma de E.coli com renaturação por alta pressão hidrostática para radiomarcação com I-131
2023 - POMIN, STEPHANIE A.
A prolactina humana é uma proteína hormonal de 23 kDa produzida principalmente pela glândula pituitária, e desempenha várias funções biológicas, como por exemplo: regulação do sistema imunológico, hematopoiese e indução da lactação. No entanto, alterações em seu nível sérico estão diretamente associadas ao desenvolvimento de tumores de mama, próstata e ovário. O objetivo desse estudo foi a produção de hPRL recombinante, contendo 6 histidinas residuais e uma metionina na região N-terminal, que pudesse ser marcada com o radioisótopo I131 para uso como uma ferramenta diagnóstica de cânceres relacionados a prolactina. Neste estudo, a hPRLhis foi produzida no citoplasma de bactérias E. coli e os corpos de inclusão gerados foram solubilizados sob alta pressão hidrostática. Foi utilizado o plasmídeo pET28a(+) com o cDNA da prolactina humana na cepa BL21(DE3). Os corpos de inclusão foram isolados e lavados. Diversas condições de solubilização foram testadas, incluindo diferentes níveis de pH e agentes redutores. A proteína foi caracterizada e sua atividade biológica foi avaliada usando células BaF/3-LLP. A radiomarcação com 131I seguiu o método de marcação direta. A prolactina humana (hPRL) foi renaturada com sucesso em condições de alta pressão (2,4 kBar) e pH 10 com incubação por 16 horas em GSSH e GSH. Cerca de 3 mg de prolactina foram obtidos a partir deste processo em um volume total de 5 mL de meio dialisado. A radiomarcação da proteína atingiu um rendimento de até 75% com 100 μCi de Iodo e 10 μg de CLT. Este estudo estabeleceu as condições para produzir hPRLhis no citoplasma bacteriano usando renaturação de alta pressão. A hPRLhis demonstrou alto nível de expressão, pureza e atividade biológica, confirmada em ensaios com células. Além disso, foi radiomarcada para futuras pesquisas com receptores em células tumorais.
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.
Comparação de diferentes meios de cultura para bactérias Escherichia coli para aumento de expressão do antagonista de prolactina humana delta 1-9 G129R-hPRL recombinante
2020 - POMIN, STEPHANIE A.; SUZUKI, MIRIAM F.
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.
Influência da temperatura na expressão do antagonista de prolactina humana delta 1-9 G129R-hPRL recombinante em bactérias Escherichia coli
2019 - POMIN, STEPHANIE A.; SUZUKI, MIRIAM F.