Preprint Article Version 2 Preserved in Portico This version is not peer-reviewed

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

Cecy Xi
,
Arianna Arianna Di Fazio
,
Naveed Nadvi
,
Karishma Patel
ORCID logo ,
Michelle Xiang
,
Hui Emma Zhang
,
Chandrika Deshpande
,
Jason Low
,
Xiaonan Trixie Wang
,
Yiqian Chen
,
Christopher McMillan
,
Ariel Isaacs
,
Brenna Osborne
,
Ana Julia Vieira de Ribeiro
,
Geoffrey McCaughan
,
Joel Mackay
ORCID logo ,
Bret Church
ORCID logo ,
Mark Gorrell
* ORCID logo
Version 1 : Received: 15 September 2020 / Approved: 17 September 2020 / Online: 17 September 2020 (08:44:17 CEST)
Version 2 : Received: 14 November 2020 / Approved: 17 November 2020 / Online: 17 November 2020 (11:38:02 CET)

A peer-reviewed article of this Preprint also exists.

Xi, C.R.; Di Fazio, A.; Nadvi, N.A.; Patel, K.; Xiang, M.S.W.; Zhang, H.E.; Deshpande, C.; Low, J.K.K.; Wang, X.T.; Chen, Y.; McMillan, C.L.D.; Isaacs, A.; Osborne, B.; Vieira de Ribeiro, A.J.; McCaughan, G.W.; Mackay, J.P.; Church, W.B.; Gorrell, M.D. A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2. Molecules 2020, 25, 5392. Xi, C.R.; Di Fazio, A.; Nadvi, N.A.; Patel, K.; Xiang, M.S.W.; Zhang, H.E.; Deshpande, C.; Low, J.K.K.; Wang, X.T.; Chen, Y.; McMillan, C.L.D.; Isaacs, A.; Osborne, B.; Vieira de Ribeiro, A.J.; McCaughan, G.W.; Mackay, J.P.; Church, W.B.; Gorrell, M.D. A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2. Molecules 2020, 25, 5392.

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 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-CoV, SARS-CoV-2 does not bind human DPP4.

Keywords

Recombinant protein; Protease; DPP4; SARS-CoV-2; MERS-CoV

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Comments (1)

Comment 1
Received: 17 November 2020
Commenter: Mark D Gorrell
Commenter's Conflict of Interests: Author
Comment: The new version adds protein structure models of the binding interfaces. We also add ELISA data that concords with our SPR data. In addition, we add ELISA and pseudovirus assay data to more clearly show, by measuring its ability to bind MERS virus and MERS spike protein, that our DPP4 is fully functional.
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