An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha.

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Item Type: Article
Status: Published
Official URL:
Journal or Publication Title: Protein Expression and Purification
Volume: 181
Page Range: p. 105833
Date: 2021
Divisions: Liver Injury and Cancer
Liver Enzymes in Metabolism and Inflammation
Gene and Stem Cell Therapy
Depositing User: General Admin
Identification Number: 10.1016/j.pep.2021.105833
ISSN: 10465928
Date Deposited: 10 Jun 2021 05:42

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.

Copyright © 2021 Elsevier Inc. All rights reserved.

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.
Last Modified: 10 Jun 2021 05:42

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