| Texto completo | |
| Autor(es): |
de Oliveira-Simoes, Flavio Antonio
;
Amatto, Isabela Victorino da Silva
;
Marciano, Camila Langer
;
Rosa-Garzon, Nathalia Gonsales da
;
Okamoto, Debora Noma
;
Juliano, Maria Aparecida
;
Juliano, Luiz
;
Cabral, Hamilton
Número total de Autores: 8
|
| Tipo de documento: | Artigo Científico |
| Fonte: | PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY; v. 54, n. 10, p. 9-pg., 2024-05-08. |
| Resumo | |
Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZ alpha A vector and purified it using immobilized metal affinity (Ni Sepharose (TM) excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain. (AU) | |
| Processo FAPESP: | 18/13588-0 - MEU concedido no processo 2012/50191-4: Espectrômetro de massas Impact II da BRUKER |
| Beneficiário: | Maria Aparecida Juliano |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa Equipamentos Multiusuários |
| Processo FAPESP: | 20/14426-3 - Clonagem, expressão e mapeamento dos subsítios de peptidases não clássicas: aplicação biotecnológica na obtenção de peptídeos bioativos |
| Beneficiário: | Hamilton Cabral |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |