RESUMO
Mass spectrometry enables the unbiased characterization of protein complexes. The success of this approach and the amount of information that can be retrieved are highly dependent on the achieved purity of the protein complex to be analyzed. Here we describe a modified tandem affinity purification (moTAP) approach which can be used to isolate the tumor necrosis factor receptor 1 signaling complex for subsequent analysis by liquid chromatography-tandem mass spectrometry. Indeed, this approach can easily be adapted to the isolation of other membrane-bound and intracellular signaling complexes. This methodology allows for a highly sensitive analysis and characterization of complex components, including posttranslational modifications and for the identification of novel complex components.
Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Complexos Multiproteicos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Reagentes de Ligações Cruzadas/química , Humanos , Complexos Multiproteicos/isolamento & purificação , Receptores Tipo I de Fatores de Necrose Tumoral/isolamento & purificaçãoRESUMO
A domain at the NH(2) terminal (N-terminal) of tumor necrosis factor receptor (TNFR) termed the pre-ligand binding assembly domain (PLAD). The finding that PLAD can mediate a selective TNFR assembly in previously researches provides a novel target to the prevention of TNFR signaling in immune-mediated inflammatory diseases (IMID). In this study, a natural N-terminal TNFR1 PLAD was obtained for the first time through the methods of GST-tag fusion protein expression and enterokinase cleavage. After purification with a Q Sepharose Fast Flow column, a natural N-terminal TNFR1 PLAD which purity was up to 95%, was obtained and was identified using Nano LC-ECI-MS/MS. Secondary structure analysis of PLAD was carried out using circular dichroism spectra (CD). After that, the TNFR1 PLAD in vitro anti-TNFα activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNFα bioactivity mainly through TNFR1. It infers an effective and safe strategy for treating variety of IMID with a low risk of side effects in future.
Assuntos
Glutationa Transferase/genética , Receptores Tipo I de Fatores de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Enteropeptidase/metabolismo , Expressão Gênica , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Tipo I de Fatores de Necrose Tumoral/isolamento & purificação , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
5His-tagged human TNFalpha type I receptor (TNFR1) ligand-binding domain was produced in Drosophila cells under control of metallothionein Cu-inducible promoter and purified by Ni-NTA affinity chromatography to homogeneity. TNFR1 gene fragment was cloned by PCR from CD8+ in vitro cultured T-killer normal linage cDNA. In despite of three disulfide bonds, the recombinant protein was correctly folded which was conformed by TNFalpha ligand binding assay in ELISA variant.
