A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Cecy R Xi; Arianna Di Fazio; Naveed Ahmed Nadvi; Karishma Patel; Michelle Sui Wen Xiang; Hui Emma Zhang; Chandrika Deshpande; Jason K K Low; Xiaonan Trixie Wang; Yiqian Chen; Christopher L D McMillan; Ariel Isaacs; Brenna Osborne; Ana Júlia Vieira de Ribeiro; Geoffrey W McCaughan; Joel P Mackay; W Bret Church; Mark D Gorrell

doi:10.3390/molecules25225392

Molecules (Nov 2020)

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Cecy R Xi,
Arianna Di Fazio,
Naveed Ahmed Nadvi,
Karishma Patel,
Michelle Sui Wen Xiang,
Hui Emma Zhang,
Chandrika Deshpande,
Jason K K Low,
Xiaonan Trixie Wang,
Yiqian Chen,
Christopher L D McMillan,
Ariel Isaacs,
Brenna Osborne,
Ana Júlia Vieira de Ribeiro,
Geoffrey W McCaughan,
Joel P Mackay,
W Bret Church,
Mark D Gorrell

Affiliations

Cecy R Xi: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Arianna Di Fazio: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Naveed Ahmed Nadvi: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Karishma Patel: Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia
Michelle Sui Wen Xiang: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Hui Emma Zhang: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Chandrika Deshpande: Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia
Jason K K Low: Faculty of Science, School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia
Xiaonan Trixie Wang: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Yiqian Chen: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Christopher L D McMillan: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia
Ariel Isaacs: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia
Brenna Osborne: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Ana Júlia Vieira de Ribeiro: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Geoffrey W McCaughan: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia
Joel P Mackay: Drug Discovery, Sydney Analytical, Core Research Facilities, The University of Sydney, Sydney, NSW 2006, Australia
W Bret Church: Faculty of Medicine and Health, School of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia
Mark D Gorrell: Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia

DOI: https://doi.org/10.3390/molecules25225392
Journal volume & issue: Vol. 25, no. 22
p. 5392

Abstract

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Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Published in Molecules

ISSN: 1420-3049 (Online)
Publisher: MDPI AG
Country of publisher: Switzerland
LCC subjects: Science: Chemistry: Organic chemistry
Website: http://www.mdpi.com/journal/molecules

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