Ni nta affinity

Plasmid pSbds-His (encoding Drosophila Sbds, amino acids 1–252, fused at the C-terminus to 6x His residues) was transformed into E. coli C41(DE3) cells and Sbds-6xHis protein were purified by Ni-NTA affinity (GE Healthcare) and a Hiload 26/60 Superdex 75 column (GE Healthcare). Protein purity was assessed by SDS-PAGE and identity confirmed by mass spectrometry.

All of the genes were cloned into pET28a with an N-terminal fusion histidine tag for expression and purification. Recombinant plasmids were first introduced into E. coli BL21 (DE3), and the bacteria were cultured in 200 mL Luria-Bertani medium at 37 °C until the OD 600 reached 0.4–0.8. 100 μM IPTG was added followed by overnight induction at 16 °C. For protein purification, the culture was centrifuged at 5000×g for 10 min at 4 °C. Then, the cells were re-suspended in buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) and centrifuged again. After ultrasonication, the re-suspended cells were centrifuged at 15000×g for 30 min at 4 °C to remove the cell debris. The supernatant was used for protein purification with a Ni-NTA affinity column and FPLC (ÄKTA, GE Healthcare Bio-Sciences) according to our previous study [25 (link)]. Finally, protein concentrations were determined using the Bradford kit (Jiancheng, Nanjing, China) according to the technical manual, and the samples were was stored at − 80 °C until use. For enzymatic reactions, different proteins (1 μg) were incubated with various substrates (approximately 1 mM) at 37 °C for 30 min (100 mM Tris-HCl pH 8.0). When necessary, 5 mM ATP, 5 mM MgC1₂, and 0.3 mM CoA were added to the reaction system according to our previous reports [25 (link), 26 (link)].

The gene of SARS-CoV-2 RBD protein and S protein with two stabilizing proline mutations at residues 986, 987 and a “GSAS” substitution at the furin cleavage site (GenBank accession no. MN908947.3) were synthesized and cloned into pCDNA3.1 expression vector using Gibson assembly [4 (link)]. Plasmids were transfected into 293F cells with Polyethyleneimine (PEI). Proteins were expressed for 5 days and supernatant was collected for further purification using Ni-NTA affinity chromatography column (GE Healthcare, Boston, MA, USA).