An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha
| Autor: | Arianna Di Fazio, W. Bret Church, Michelle Sui Wen Xiang, Xin Maggie Wang, Cecy R. Xi, Hui Emma Zhang, Charles G. Bailey, Geoffrey W. McCaughan, Chandrika N. Deshpande, Yiqian Chen, Naveed A. Nadvi, Mehdi Sharifi Tabar, Mark D. Gorrell |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
congenital hereditary and neonatal diseases and abnormalities medicine.medical_treatment Recombinant Fusion Proteins Spodoptera 01 natural sciences law.invention 03 medical and health sciences Protein structure Fibroblast activation protein alpha law 010608 biotechnology Endopeptidases medicine Sf9 Cells Animals Humans neoplasms 030304 developmental biology chemistry.chemical_classification Gel electrophoresis 0303 health sciences Protease Membrane Proteins digestive system diseases Transmembrane protein Transmembrane domain chemistry Biochemistry Recombinant DNA Glycoprotein Biotechnology |
| Zdroj: | Protein expression and purification. 181 |
| ISSN: | 1096-0279 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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