E-Poster with pre-recorded video presentation Lorne Infection and Immunity 2021

A novel purification procedure for active recombinant human DPP4 and the inability of DPP4 to bind SARS-CoV-2 (#207)

Cecy R Xi 1 2 , Arianna Di Fazio 1 2 , Naveed Ahmed Nadvi 1 2 , Karishma Patel 2 , Michelle SW Xiang 1 , Hui Emma Zhang 1 2 , Chandrika Deshpande 2 , Jason KK Low 2 , Xiaonan Trixie Wang 1 , Yiqian Chen 1 2 , Christopher LD McMillan 3 , Ariel Isaacs 3 , Brenna Osborne 1 , Ana Júlia Vieira de Ribeiro 1 , Geoffrey W McCaughan 1 2 4 , Joel P Mackay 2 , W Bret Church 2 , Mark D Gorrell 1 2
  1. Centenary Institute, Camperdown, NSW, Australia
  2. The University of Sydney, Sydney
  3. The University of Queensland, St Lucia, QLD, Australia
  4. AW Morrow GE & Liver Centre, Royal Prince Alfred Hospital, Camperdown, NSW, Australia

 

The protease, dipeptidyl peptidase 4 (DPP4), is a pharmacological target in type 2 diabetes therapy. 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. The current study aimed to develop such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced from Spodoptera frugiperda  9 (Sf9) insect cells.  Purification used differential ammonium sulfate 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 using 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. This DPP4 bound to MERS-CoV spike. No specific binding between DPP4 and CoV-2 spike or spike RBD was detected. 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.