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Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain.
de Oliveira-Simões, Flávio Antônio; Victorino da Silva Amatto, Isabela; Langer Marciano, Camila; Rosa-Garzon, Nathalia Gonsales da; Noma Okamoto, Débora; Juliano, Maria Aparecida; Juliano, Luiz; Cabral, Hamilton.
Afiliación
  • de Oliveira-Simões FA; Pharmaceutical Sciences Program, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
  • Victorino da Silva Amatto I; Biosciences and Biotechnology Program, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
  • Langer Marciano C; Biosciences and Biotechnology Program, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
  • Rosa-Garzon NGD; Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
  • Noma Okamoto D; Deparatment of Pharmaceutical Sciences, Universidade Federal de São Paulo, Diadema, SP, Brazil.
  • Juliano MA; Departament of Biophysical, Escola Paulista de Medicina, Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
  • Juliano L; Departament of Biophysical, Escola Paulista de Medicina, Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
  • Cabral H; Pharmaceutical Sciences Program, Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Prep Biochem Biotechnol ; : 1-9, 2024 May 10.
Article en En | MEDLINE | ID: mdl-38727020
ABSTRACT
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 pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Prep Biochem Biotechnol Asunto de la revista: BIOQUIMICA / BIOTECNOLOGIA Año: 2024 Tipo del documento: Article País de afiliación: Brasil Pais de publicación: Reino Unido
Texto completo
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Prep Biochem Biotechnol Asunto de la revista: BIOQUIMICA / BIOTECNOLOGIA Año: 2024 Tipo del documento: Article País de afiliación: Brasil Pais de publicación: Reino Unido