Materials and Methods
For NMR study, a double-stranded DNA fragment encoding N-terminal NH-tagged SARS-CoV-2 proteins was synthesized (Thermo Fisher Scientific), with a HRV3C protease sequence located between NH-tag and the target protein. The DNA fragment was subjected to a 16-hr in vitro transcription and translation with stable isotope labeled amino acids for a ILVM methyl 1H13C-labeling under a 2H15N-labeling background. Cell-Free Protein Expression Kit and Stable Isotope Labeled Amino Acids (see below) are obtained from Taiyo Nippon Sanso Corp. (Ref. 2). Expression conditions of each protein are summarized in Table I.
The resultant reaction solution containing NH-tagged SARS-CoV-2 proteins was purified by Ni-NTA resin (WAKO) which was equilibrated with a buffer containing 20 mM Tris-HCl (pH8.0), 300 mM NaCl. The resin was washed with the same buffer with 10 mM imidazole, followed by elution with the buffer with 300 mM imidazole.
The purified SARS-CoV-2 proteins were subjected to buffer exchange with the NMR buffer (20 mM deuterated HEPES (pH 7.0), 150 mM NaCl, 90% H2O, 10% D2O).
Fore those expressed with DTT, 2 mM DTT were added to NMR sample.
For those expressed with the Membrane Protein Expression Additive M1 (Taiyo Nippon
Sanso Corp.), 0.01 % DDM were added to NMR sample.
NMR experiments were performed using a Bruker Avance-III 600 MHz or 800 MHz spectrometer equipped with cryogenic triple resonance probes (Bruker) at 298K. TOPSPIN (Bruker) was used to process NMR spectra.
Protein Name | Length (a.a.) |
Expression region (from N-term of each protein) |
Protein/Domain type | Expression Conditions | NMR | |||||
---|---|---|---|---|---|---|---|---|---|---|
PEG | DTT | C4 | M1 | Zn | 13C | 15N | ||||
Nsp1 FL | 180 | 1-180 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp3 1-112 | 112 | 1-112 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp3 205-379 | 175 | 205-379 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp3 401-530 | 130 | 401-530 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp3 1088-1203 | 116 | 1088-1203 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp4 401-500 | 100 | 401-500 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp5 FL | 306 | 306 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp7 FL | 83 | 1-83 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp8 FL | 198 | 1-198 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp9 FL | 113 | 1-113 | Intracellular Protein | ○ | ○ | - | - | - | ● | ● |
Nsp16 FL | 298 | 1-298 | Intracellular Protein | ○ | ○ | - | - | - | ● | |
Nucleocapsid protein, NTD | 134 | 41-174 | Intracellular Domain | ○ | ○ | - | - | - | ● | ● |
Nucleoprotein, CTD | 118 | 247-364 | Intracellular Domain | ○ | ○ | - | - | - | ● | ● |
ORF9b | 105 | 1-105 | Intracellular Protein | ○ | ○ | - | - | - | ● | |
Nsp10 FL | 139 | 1-139 | Intracellular Protein/DNA binding | ○ | ○ | - | - | ○ | ● | |
Nsp15 FL | 346 | 1-346 | Intracellular Protein/DNA binding | ○ | ○ | - | - | ○ | ● | ● |
S 330-532 | 203 | 330-532 | Extracellular Domain | ○ | - | ○ | - | - | ● | ● |
S 330-532 E484K | 203 | 330-532 | Extracellular Domain | ○ | - | ○ | - | - | ● | ● |
S 330-532 N501Y | 203 | 330-532 | Extracellular Domain | ○ | - | ○ | - | - | ● | ● |
ORF7a 16-82 | 67 | 16-82 | Extracellular Domain | ○ | - | ○ | - | - | ● | ● |
Orf3b | 22 | 1-22 | Putative membrane protein | - | ○ | - | ○ | - | ● | |
ORF4a FL | 75 | 1-75 | Putative membrane protein | - | ○ | - | ○ | - | ● | |
ORF6a FL | 61 | 1-61 | Putative membrane protein | - | ○ | - | ○ | - | ● | |
ORF10 FL | 38 | 1-38 | Putative membrane protein | - | ○ | - | ○ | - | ● | |
Non-Canonical NC128 | 51 | 1-51 | Putative membrane protein | - | ○ | - | ○ | - | ● |
Orf3b | |||||
---|---|---|---|---|---|
10 | 20 | ||||
MMPTIFFAGI | LIVTTIVYLT | IV | |||
Orf9b | |||||
10 | 20 | 30 | 40 | 50 | |
MNKLKCLIMD | PKISEMHPAL | RLVDPQIQLA | VTRMENAVGR | DQNNVGPKVY | |
60 | 70 | 80 | 90 | 100 | |
PIILRLGSPL | SLNMARKTLN | SLEDKAFQLT | PIAVQMTKLA | TTEELPDEFV | VVTVK |
NC-128 | |||||
10 | 20 | 30 | 40 | 50 | |
MIRKSKLVLK | KLQQLWKKLS | SSQKTCYFIL | TLMAIFIQIL | PLLLVTLTSL | S |