The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding

Courtney Mycroft-West; Dunhao Su; Stefano Elli; Scott Guimond; Gavin Miller; Jeremy Turnbull; Edwin Yates; Marco Guerrini; David Fernig; Marcelo Lima; Mark Skidmore

Residues 330−583 of the SARS-CoV-2 Spike Protein (GenBank: MN908947) were cloned upstream of a N-terminal 6XHisTag in the pRSETA expression vector and transformed into SHuffle® T7 Express Competent E. coli (NEB, UK). Protein expression was carried out in MagicMedia™ E. coli Expression Media (Invitrogen, UK) at 30°C for 24 hrs, 250 rpm. The bacterial pellet was suspended in 5 mL lysis buffer (BugBuster Protein Extraction Reagent, Merck Millipore, UK; containing DNAse) and incubated at room temperature for 30 mins. Protein was purified from inclusion bodies using IMAC chromatography under denaturing conditions. On-column protein refolding was performed by applying a gradient with decreasing concentrations of the denaturing agent (6-0 M Urea). After extensive washing, protein was eluted using 20 mM NaH2PO4, pH 8.0, 300 mM NaCl, 500 mM imidazole. Fractions were pooled and buffer-exchanged to phosphate-buffered saline (PBS; 140 mM NaCl, 5 mM NaH2PO4, 5 mM Na2HPO4, pH 7.4; Lonza, UK) using a PD-10 desalting column (GE Healthcare, UK). Recombinant protein was stored at -20°C until required. the author/funder. All rights reserved. No reuse allowed without permission.