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

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

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Item Type: Article
Status: Published
Official URL: https://doi.org/10.3390/molecules25225392
Journal or Publication Title: Molecules
Volume: 25
Number: 22
Page Range: p. 5392
Date: 2020
Divisions: Liver Enzymes in Metabolism and Inflammation
Liver Injury and Cancer
Depositing User: General Admin
Identification Number: 10.3390/molecules25225392
ISSN: 1420-3049
Date Deposited: 05 Jan 2021 05:15
Abstract:

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.

Creators:
Creators
Email
Xi, Cecy R
UNSPECIFIED
Di Fazio, Arianna
UNSPECIFIED
Nadvi, Naveed Ahmed
UNSPECIFIED
Patel, Karishma
UNSPECIFIED
Xiang, Michelle Sui Wen
UNSPECIFIED
Zhang, Hui Emma
UNSPECIFIED
Deshpande, Chandrika
UNSPECIFIED
Low, Jason K K
UNSPECIFIED
Wang, Xiaonan Trixie
UNSPECIFIED
Chen, Yiqian
UNSPECIFIED
McMillan, Christopher L D
UNSPECIFIED
Isaacs, Ariel
UNSPECIFIED
Osborne, Brenna
UNSPECIFIED
Vieira de Ribeiro, Ana Júlia
UNSPECIFIED
McCaughan, Geoffrey W
UNSPECIFIED
Mackay, Joel P
UNSPECIFIED
Church, W Bret
UNSPECIFIED
Gorrell, Mark D
UNSPECIFIED
Last Modified: 05 Jan 2021 05:15
URI: https://eprints.centenary.org.au/id/eprint/920

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