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

Cecy R Xi; Arianna Di Fazio; Naveed Ahmed Nadvi; Michelle Sui Wen Xiang; Hui Emma Zhang; Chandrika Deshpande; Yiqian Chen; Mehdi Sharifi Tabar; Xin Maggie Wang; Charles G Bailey; Geoffrey W McCaughan; W Bret Church; Mark D Gorrell

doi:10.1016/j.pep.2021.105833

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

Protein Expr Purif. 2021 May:181:105833. doi: 10.1016/j.pep.2021.105833. Epub 2021 Jan 30.

Affiliations

¹ Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
² 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.
³ 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.
⁴ Gene & Stem Cell Therapy Program, Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia.
⁵ 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.
⁶ Faculty of Medicine and Health, School of Pharmacy, The University of Sydney, Sydney, New South Wales, 2006, Australia.
⁷ Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, 2006, Australia. Electronic address: m.gorrell@centenary.org.au.

PMID: 33524496
DOI: 10.1016/j.pep.2021.105833

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.

Keywords: Baculovirus; Fibroblast activation protein alpha; Protease; Recombinant protein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Endopeptidases* / biosynthesis
Endopeptidases* / chemistry
Endopeptidases* / genetics
Endopeptidases* / isolation & purification
Humans
Membrane Proteins* / biosynthesis
Membrane Proteins* / chemistry
Membrane Proteins* / genetics
Membrane Proteins* / isolation & purification
Recombinant Fusion Proteins / biosynthesis
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / isolation & purification
Sf9 Cells
Spodoptera

Substances

Membrane Proteins
Recombinant Fusion Proteins
Endopeptidases
fibroblast activation protein alpha