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

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.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:25

Enthalten in:

Molecules (Basel, Switzerland) - 25(2020), 22 vom: 18. Nov.

Sprache:

Englisch

Beteiligte Personen:

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

Links:

Volltext

Themen:

ACE2 protein, human
Angiotensin-Converting Enzyme 2
Covid-19
DPP4
DPP4 protein, human
Dipeptidyl Peptidase 4
EC 3.4.14.5
EC 3.4.17.23
Journal Article
Protease
Recombinant Proteins
Recombinant protein
Spike Glycoprotein, Coronavirus
Spike protein, SARS-CoV-2

Anmerkungen:

Date Completed 04.12.2020

Date Revised 14.12.2020

published: Electronic

Citation Status MEDLINE

doi:

10.3390/molecules25225392

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM317850733