Structural Characterization of Bat Coronavirus Proteins

The gene encoding M protein of Pipistrellus bat coronavirus HKU5 (batCOV5-M, NCBI accession YP_001039968.1) was synthesized (Genewiz, China) and cloned into a modified pPICZ plasmid (Thermo Fisher Scientific) containing an N-terminal tag of FLAG-His₁₀-TEV protease recognition site. All batCOV5-M mutations were introduced by QuikChange II system (Agilent) according to manufacturer's recommendation, and all mutations were verified by DNA sequencing. The constructs were linearized and transformed into Pichia pastoris strain GS115 by lithium chloride/single-strand carrier DNA/polyethylene glycol method according to manufacturer's manual (Thermo Fisher Scientific). The transformants were inoculated into YPD medium consisting of 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) D-(+)-glucose at 30°C with shaking at 220 rpm until an OD₆₀₀ of 3–5 was reached. To induce protein expression, yeast cells were harvested by centrifugation and resuspended to an OD₆₀₀ of 1 in YPM medium consisting of 1% (w/v) yeast extract, 2% (w/v) peptone, 0.8% (v/v) methanol, and 2.5% (v/v) dimethyl sulfoxide at 30°C for 24 h. Cell pellets were resuspended in Lysis Solution (LS) containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 mM β-mercaptoethanol, and were lysed by an AH-1500 high-pressure homogenizer (ATS, China) at 1,300 MPa. Undisrupted cells and cell debris were separated by centrifugation at 3,000×g, and membrane was collected by ultracentrifugation at 140,000×g for 1 h at 4°C. Protein was extracted by addition of 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM; Anatrace) at 4°C for 2 h and the extraction mixture was centrifuged at 200,000×g for 30 min at 4°C. The supernatant was incubated with Co²⁺ resin in the presence of 20 mM imidazole pH 8.0 at 4°C for 1 h, and the mixture was loaded in an empty chromatography column. The resin/protein was washed with 20 bed-volume of LS containing 2 mM DDM and 30 mM imidazole pH 8.0, and the protein was eluted with LS supplemented with 2 mM DDM and 250 mM imidazole pH 8.0.
To generate the GFP-batCOV5-M_CTD construct, the DNA sequence encoding batCOV5-M_CTD (residues 115–203 of batCOV5-M) was amplified by polymerase chain reaction (PCR) and cloned into a modified pPICZ plasmid (Thermo Fisher Scientific) containing a N-terminal His₁₀-TEV site-GFP tag. To generate the batCOV5-M_CTD-sfGFP construct, a superfolder GFP (35 (link)) was split into two halves (21 (link)) and was fused to the N- and C-termini of batCOV5-M_CTD (residues 115–203 of batCOV5-M) by gene synthesis (Genewiz, China). The fusion protein-encoding DNA was cloned into a modified pPICZ plasmid (Thermo Fisher Scientific) containing a C-terminal TEV site and a His₁₀ tag. Transformation and expression of GFP-batCOV5-M_CTD and batCOV5-M_CTD-sfGFP followed the same protocol as batCOV5-M except that the expression was induced at 25°C. Cell pellets were resuspended in LS containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 20% (v/v) glycerol and 1 mM PMSF, and were lysed similarly to batCOV5-M. Undisrupted cells and cell debris were separated by centrifugation at 140,000×g at 4°C for 1 h. The supernatant was supplemented with 20 mM imidazole pH 8.0 and was immediately loaded onto a pre-washed Co²⁺ affinity column. The column was then washed with 20 bed-volume of LS containing 30 mM imidazole pH 8.0, and the protein was eluted with LS containing 250 mM imidazole pH 8.0.
The gene encoding N protein of batCOV5 (batCOV5-N, NCBI accession YP_001039969.1) was synthesized (Genewiz, China) and cloned into a modified pPICZ plasmid (Thermo Fisher Scientific) containing an N-terminal tag of FLAG-His₁₀-TEV site, followed by the bacterial cytochrome b562RIL (BRIL) (48 (link)) to improve expression of the protein of interest. All batCOV5-N fragments were generated by PCR and cloned into the same vector as batCOV5-N. For MST analysis, batCOV5-N₃-enconding DNA was subcloned into another modified pPICZ plasmid (Thermo Fisher Scientific) containing a C-terminal tag of TEV site-GFP-His₁₀. Single mutations of batCOV5-N₃ were introduced by QuikChange II system (Agilent). Transformation, expression, and purification of batCOV5-N and fragments followed the same protocol as batCOV5-M_CTD-sfGFP, except that LS contained 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol and 1 mM PMSF.