Recombinant peptide n glycosidase f pngase f

Clinical specimens collected from infected patients containing SARS-CoV-2 were grown in cell culture using Vero E6 cells following a reported procedure [21 ] and, as used in a previous study [12 (link)], were the source of an originating-like strain. Following chemical and heat inactivation and filtration, the virus was precipitated with polyethylene glycol precipitation of virus was performed after filtration through a 300-K molecular weight cut-off (MWCO) filter (Pall Corporation, Cheltenham, Victoria). The retentate was reconstituted in buffer (50 mM ammonium bicarbonate), sonicated (3 × 30 min) and then deglycosylated following the addition of 1.2 units of recombinant peptide-N-glycosidase F (PNGaseF) (Roche Diagnostics, North Ryde, Sydney, Australia) and 5 mM octyl β-D-glucopyranoside (Sigma Aldrich–Merck, Castle Hill, Sydney, Australia). The released viral proteins were separated by SDS-PAGE and the S-protein (at some 150 kDa) was excised from the gel. The gel plug was transferred into 25 mM ammonium bicarbonate solution containing 10% v/v acetonitrile (ACN) and 10 mM dithiothreitol (DTT) (10 mM) and heated for 30 min at 60 °C. The gel plug was washed three times with 25 mM ammonium bicarbonate in 50% acetonitrile and then dried in a vacuum concentrator (Labconco Corporation, Kansas City, MI, USA).

Analytical grade ethanol,
sodium dodecyl sulfate (SDS), trifluoroacetic acid (TFA), and potassium
hydroxide (KOH) were obtained from Merck (Darmstadt, Germany). Disodium
hydrogen phosphate dihydrate (Na₂HPO₄ ×
2H₂O), potassium dihydrogen phosphate (KH₂PO₄), sodium chloride (NaCl), N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC), 85% phosphoric
acid (H₃PO₄), 50% sodium hydroxide (NaOH), nonidet
P-40 substitute (NP-40), 1-hydroxybenzotriazole 97% (HOBt) and super-DHB
(9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic
acid, sDHB) were purchased from Sigma-Aldrich (Steinheim, Germany).
1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) hydrochloride
was obtained from Fluorochem (Hadfield, UK), whereas recombinant peptide-N-glycosidase F (PNGase F) was obtained from Roche Diagnostics
(Mannheim, Germany) and HPLC-grade acetonitrile (ACN) was purchased
from Biosolve (Valkenswaard, The Netherlands). Milli-Q water (MQ)
was generated from a Q-Gard 2 system (Millipore, Amsterdam, The Netherlands),
which was maintained at ≥18 MΩ.