Your browser doesn't support javascript.
loading
New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept.
Contreras, María Angélica; Macaya, Luis; Neira, Pedro; Camacho, Frank; González, Alaín; Acosta, Jannel; Montesino, Raquel; Toledo, Jorge Roberto; Sánchez, Oliberto.
Afiliação
  • Contreras MA; Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • Macaya L; Center for Biotechnology and Biomedicine Spa, Concepción, Chile.
  • Neira P; Center for Biotechnology and Biomedicine Spa, Concepción, Chile.
  • Camacho F; Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • González A; Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • Acosta J; Recombinant Biopharmaceuticals Laboratory, Pharmacology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • Montesino R; Biotechnology and Biopharmaceutical Laboratory, Pathophysiology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • Toledo JR; Biotechnology and Biopharmaceutical Laboratory, Pathophysiology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, PO Box 160-C, Concepción, Chile.
  • Sánchez O; Center for Biotechnology and Biomedicine Spa, Concepción, Chile.
Biochem J ; 477(17): 3299-3311, 2020 09 18.
Article em En | MEDLINE | ID: mdl-32941643
TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9 M and >10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.
Assuntos
Palavras-chave
Texto completo
Adicionar na Minha BVS
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fator de Necrose Tumoral alfa / Adalimumab / Etanercepte Limite: Humans Idioma: En Revista: Biochem J Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Chile País de publicação: Reino Unido
Texto completo
Adicionar na Minha BVS
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Fator de Necrose Tumoral alfa / Adalimumab / Etanercepte Limite: Humans Idioma: En Revista: Biochem J Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Chile País de publicação: Reino Unido