Protocol for production and characterization of SARS-CoV-2 PL(pro) in Escherichia coli.
De Falco A, Greene-Cramer R, Shurina BA, Zakian S, Acton TB, Ramelot TA, Montelione GT
STAR Protoc. 2025 Sep 19;6(3):103952. doi: 10.1016/j.xpro.2025.103952. Epub 2025 Jul 18.
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Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 233739 | pGTM_COV2_NSP3_003_SUMO | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of an active PLpro protease. Following Sumo Protease cleavage no nonnative residues remain on the PLpro protein |
| 233740 | pGTM_SUMO_PLpro_C111S | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of PLpro protease with a C111S active site mutation that abolishes protease activity. Following Sumo Protease cleavage no nonnative res |
| 233803 | pGTM_Sumo2-N-8XHis-Plpro | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of an active PLpro protease. Following Sumo Protease Cleavage an 8xHis Tag is retained at the N-terminus |
| 234321 | pGTM_Sumo2-C-8XHis-PLpro | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of an active PLpro protease. Following Sumo Protease Cleavage an 8xHis Tag is retained at the C-terminus |
| 234322 | pGTM_Sumo2-N-StrepII-PLpro | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of an active PLpro protease. Following Sumo Protease Cleavage a Strep-tag II sequence is retained at the N-terminus |
| 234472 | pGTM_Sumo2-N-8XHis-Plpro-C111S | Expresses SARS-CoV-2 Nsp3 residues 746-1063 C111S with an activity abolishing mutation for the purification of this PLpro protease. Following Sumo Protease Cleavage an N-term 8xHis Tag is retained |
| 234489 | pGTM_Sumo_PLpro_C111A | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of PLpro protease with a C111A active site mutation that abolishes protease activity. Following Sumo Protease cleavage no nonnative res |
| 234490 | pGTM_SUMO_PLpro_W93F_001 | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of PLpro protease with a W93F mutation to allow for 5-fluorotryptophan incorporation and 19F NMR |
| 234491 | pGTM_SUMO_PLpro_W106F_001 | Expresses SARS-CoV-2 Nsp3 residues 746-1063 for the purification of PLpro protease with a W106F mutation to allow for 5-fluorotryptophan incorporation and 19F NMR |