Hiload 16 600 superdex 200 column

A plasmid corresponding to the semi-stabilized SARS-CoV-2 S-2P protein^{9 (link)} was synthesized, codon-optimized, cloned into pCDNA2004, and sequenced at GenScript (Piscataway, NJ 08854), where also all the variants with different amino acid substitutions were generated. A variant with a HIS-tag and a variant with a C-tag were purified. The expression platform used was the Expi293F cells. The cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 6 days at 37 °C and 10% CO₂. The culture supernatants were harvested and spun for 5 minutes at 300 × g to remove cells and cellular debris. The spun supernatants were subsequently sterile filtered using a 0.22 μm vacuum filter and stored at 4 °C until they were purified within 1–2 days of harvest. HIS-tagged SARS-CoV-2 S trimers were purified using a two-step purification protocol by 1- or 5-ml complete HIS-tag columns (Roche). Proteins were further purified by size-exclusion chromatography using a HiLoad Superdex 200 16/600 column (GE Healthcare).

CAMKK2 kinase domain (132–470) was cloned in a pNIC-Bio2 vector in fusion with N-terminal 10xHis tag followed by a TEV protease cleavage site and a C-terminal biotin ligase recognition sequence. This construct was used in the expression of CAMKK2 in E. coli BL21(DE3)-R3-BirA [44 (link)]. Protein was purified in a Ni-NTA column (Thermo Scientific, Waltham, MA, USA) followed TEV digestion overnight, dialysis to remove imidazole and re-purification in Ni-NTA to remove undigested samples and TEV protease (made in house with an N-terminal 6xHis tag). As a last step, this sample was loaded to a HiLoad Superdex 200 16/600 column (GE Healthcare, Chicago, IL, USA) for final polishing and buffer exchange.
Tracer displacement assay was measured in 15 µL volume containing 5 nM of our C-terminal biotinylated CAMKK2 kinase domain, 2 nM of Europium-labeled streptavidin in buffer 50 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35 and 8 nM of tracer 236 (measured K_D of 8.13 ± 0.9 nM) as described [45 ].

The genes coding for FliD_cj, FliD_sm, and FliD_pa, codon-optimized for expression in E. coli, were synthesized (BioBasic) and sub-cloned into pET28a (Novagen) (Supplementary Table 4). Recombinant proteins were expressed in E. coli BL21-CodonPlus(DE3)-RIL cells containing the corresponding plasmids. For FliD_cj, transformants were grown in LB medium at 37 °C until they reached log phase, and expression was induced by the addition of 1 mM IPTG overnight at 20 °C. For both FliD_pa and FliD_sm, expression was auto-induced in ZYM-5052^{33 (link)} media at 20 °C overnight. For all three proteins, cells were collected by centrifugation, resuspended in 50 mM HEPES 150 mM NaCl pH 7 and sonicated. The lysate was centrifuged at 14,000g at 4 °C for 45 min. The supernatants were applied onto a 5 ml HisPure™ Ni-NTA resin (ThermoScientific) gravity-based column equilibrated with 50 mM HEPES 150 mM NaCl pH 7 and eluted using a linear 20–500 mM Imidazole gradient. Fractions containing FliD were pooled and applied to a HiLoad Superdex 200 16/600 column (GE Healthcare) equilibrated with 50 mM HEPES 150 mM NaCl pH 7 for FliD_cj and 50 mM Tris 150 mM NaCl pH 8 for FliD_pa and FliD_sm.