Strep tactin sepharose beads

Recombinant trimeric IAV hemagglutinin proteins open reading frames were cloned into the pCD5 expression vector as described previously^{55 (link)}, in frame with a GCN4 trimerization motif (KQIEDKIEEIESKQKKIENEIARIKK), a superfolder GFP^{56 (link)} and the Twin-Strep-tag (WSHPQFEKGGGSGGGSWSHPQFEK); (IBA, Germany). The open reading frames of the HAs of A/Hong-Kong/1/1968 H3 (AFG71887.1), A/Netherlands/816/1991 H3 (EPI_ISL_114608), A/Netherlands/109/2003 H3 (EPI_ISL_113016), A/Netherlands/761/2009 H3 (EPI_ISL_1107270), A/Singapore/INFH-16-0019/2016 H3 (3 C.2a) (QQY97257.1), were synthesized and codon optimized by GenScript.
The trimeric HAs were expressed in HEK293T (CRL-11268) and HEK 293 S GNTI(-) (ATCC CRL-3022) cells with polyethyleneimine I (PEI) in a 1:8 ratio (µg DNA:µg PEI) for the HAs as previously described^{16 (link)}. The transfection mix was replaced after 6 h by 293 SFM II suspension medium (Invitrogen, 11686029), supplemented with sodium bicarbonate (3.7 g/L), Primatone RL-UF (3.0 g/L, Kerry, NY, USA), glucose (2.0 g/L), glutaMAX (1%, Gibco), valproic acid (0.4 g/L) and DMSO (1.5%). Culture supernatants were harvested 5 days post-transfection and the HA0 was purified with sepharose strep-tactin beads (IBA Life Sciences, Germany) according to the manufacturer’s instructions.

At 48 h post-transfection, cells were dissociated from the plate surface with 1× phosphate-buffered saline (PBS) containing 10 mM EDTA, subsequently washed with cold 1× PBS, and lysed in IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Nonidet P40 substitute (NP-40; Solarbio, Beijing, China) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche, Bransburg, NJ, USA). Cells were lysed on ice for 30 min then cleared by centrifugation at 17,000× g for 10 min at 4 °C. After centrifugation, the supernatant was incubated with 30 μL Strep-Tactin Sepharose beads (IBA Lifesciences, Göttingen, Germany) diluted in IP Buffer for 2 h. Beads were then washed three times with 1 mL IP buffer supplemented with 0.05% NP-40 and transferred to a new tube with a final wash in 1 mL IP buffer. Proteins were eluted by agitating beads in 40 μL IP buffer supplemented with 2.5 mM D-desthiobiotin (IBA Lifesciences) on a vortex mixer at room temperature for 30 min. We reserved 10% of each eluate for western blotting and silver staining. The remaining eluate was removed for mass spectrometry (MS).

Selected CaMKIIβ isoforms were expressed in High Five insect cells via the baculovirus system. All purification steps were performed at 4°C. Cell pellets were resuspended in CaMKII lysis buffer (10 mM Tris–HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, and 1 mM DTT) supplemented with protease inhibitors (cOmplete; Roche) and lysed by sonication. Insoluble particles were separated by centrifugation at 21,500 rpm for 1 h (JA-25.50 fixed-angle rotor; Beckman Coulter). The soluble fraction was incubated with Strep-Tactin Sepharose beads (IBA Lifesciences) for 1 h and washed with CaMKII lysis buffer. Bound protein was eluted with CaMKII SEC buffer (50 mM Pipes, pH 7.5, 500 mM NaCl, 1 mM EGTA, 10% glycerol, and 1 mM DTT) containing 2.5 mM desthiobiotin (IBA Lifesciences). Eluted protein was concentrated and run on a Superose 6 10/300 Gl size-exclusion column (Cytiva) with CaMKII SEC buffer. Fractions were pooled according to SDS–PAGE and chromatogram, concentrated to ∼1 mg/ml, and flash-frozen in single-use aliquots in liquid nitrogen. Before use, aliquots were thawed on ice, gently mixed by pipetting, and centrifuged at 20,000 rcf for 5 min. Exactly equal concentrations were determined by repeated SDS–PAGE, Coomassie staining, and quantification with ImageQuant TL (Cytiva).

To verify that the 2 betacoronavirus infections in dogs (SARS-CoV-2 and canine respiratory coronavirus [CRCoV]) can be distinguished serologically, we designed an antigen S1 adsorption assay. We incubated serum samples with Strep-Tactin Sepharose Beads (IBA Lifesciences, https://www.iba-lifesciences.com) conjugated with S1 protein of SARS-CoV-2, BCoV, or HCoV-229E and titrated mock-absorbed and protein-absorbed serum samples in the ELISA. We expressed IgG titers as the reciprocal of highest serum dilution resulting in OD values above the cutoff value.