Protocol for production and characterization of SARS-CoV-2 PL in Escherichia coli.

Publication date: Jul 18, 2025

Papain-like protease (PL) from SARS-CoV-2 is a high-priority target for COVID-19 antiviral drug development. Here, we present a protocol for PL production in Escherichia coli. We describe steps for expressing PL as a fusion with a SUMO tag (small ubiquitin-like modifier) and purifying and obtaining it in its native form upon hydrolysis, with good yields. We provide approaches for PL characterization by mass spectrometry, NMR, and a fluorescence-based enzyme assay. We also detail a technique for producing isotope-enriched samples suitable for NMR studies.

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Concepts	Keywords
Coli	biotechnology and bioengineering
Covid	NMR
Hydrolysis
Spectrometry
Sumo

Semantics

Type	Source	Name
disease	IDO	production
drug	DRUGBANK	Papain
disease	MESH	COVID-19
disease	IDO	assay
drug	DRUGBANK	Ademetionine
pathway	KEGG	Viral replication
disease	IDO	host
drug	DRUGBANK	L-Tryptophan
disease	IDO	protein
disease	IDO	site
disease	IDO	replication
disease	MESH	ampicillin resistance
disease	MESH	tryptophan
disease	IDO	reagent
drug	DRUGBANK	Ampicillin
drug	DRUGBANK	Ethanol
drug	DRUGBANK	Isopropyl beta-D-thiogalactopyranoside
drug	DRUGBANK	Water
drug	DRUGBANK	Dimethyl sulfoxide
drug	DRUGBANK	Coenzyme M
drug	DRUGBANK	Activated charcoal
drug	DRUGBANK	Methylergometrine
drug	DRUGBANK	Sodium Chloride
drug	DRUGBANK	Tromethamine
drug	DRUGBANK	Imidazole
drug	DRUGBANK	Zinc chloride
drug	DRUGBANK	Gold
drug	DRUGBANK	Chloride ion
drug	DRUGBANK	Zinc sulfate
drug	DRUGBANK	Sodium molybdate
drug	DRUGBANK	Sulfate ion
drug	DRUGBANK	Boric acid
drug	DRUGBANK	Sodium phosphate monobasic
drug	DRUGBANK	Monopotassium phosphate
drug	DRUGBANK	Dipotassium phosphate
drug	DRUGBANK	Acetylcysteine
drug	DRUGBANK	Magnesium sulfate
disease	IDO	process
drug	DRUGBANK	Sodium lauryl sulfate
disease	MESH	shock
disease	IDO	colony
drug	DRUGBANK	Iron
drug	DRUGBANK	Nitrogen
drug	DRUGBANK	Glycerin
drug	DRUGBANK	Formic Acid
drug	DRUGBANK	Medical air
drug	DRUGBANK	L-Arginine
drug	DRUGBANK	L-Leucine
drug	DRUGBANK	Glycine
drug	DRUGBANK	Acetate ion
drug	DRUGBANK	L-Cysteine
disease	IDO	cell
drug	DRUGBANK	Proline

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