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An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.
Xi, Cecy R; Di Fazio, Arianna; Nadvi, Naveed Ahmed; Xiang, Michelle Sui Wen; Zhang, Hui Emma; Deshpande, Chandrika; Chen, Yiqian; Tabar, Mehdi Sharifi; Wang, Xin Maggie; Bailey, Charles G; McCaughan, Geoffrey W; Church, W Bret; Gorrell, Mark D.
Afiliación
  • Xi CR; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Di Fazio A; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Nadvi NA; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia; Research Portfolio Core Research Facilities, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Xiang MSW; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Zhang HE; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Deshpande C; Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, 2006, UK; Drug Discovery, Sydney Analytical, Core Research Facilities, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Chen Y; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Tabar MS; Gene & Stem Cell Therapy Program, Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Wang XM; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Bailey CG; Gene & Stem Cell Therapy Program, Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • McCaughan GW; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia; AW Morrow GE & Liver Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, 2050, Australia.
  • Church WB; Faculty of Medicine and Health, School of Pharmacy, The University of Sydney, Sydney, New South Wales, 2006, Australia.
  • Gorrell MD; Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia. Electronic address: m.gorrell@centenary.org.au.
Protein Expr Purif ; 181: 105833, 2021 05.
Article en En | MEDLINE | ID: mdl-33524496
ABSTRACT
Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27-760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 µM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Endopeptidasas / Proteínas de la Membrana Límite: Animals / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2021 Tipo del documento: Article País de afiliación: Australia
Texto completo
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Imprimir
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Endopeptidasas / Proteínas de la Membrana Límite: Animals / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2021 Tipo del documento: Article País de afiliación: Australia