Table 4.

Commonly used Proteases to Remove Fusion Tags from Recombinant Proteins

Protease	Description	Cleavage sitea	Protease inhibitors	Activity	Comments
TEV	Catalytic domain of the Nuclear Inclusion a (NIa) protein, a cysteine protease, found in the tobacco etch virus (TEV)	EXXYXQ‐(G/S) Most common: ENLYFQ‐X	PMSF, AEBSF, TLCK, pepstatin A, bestatin, E‐64, zinc (>5 mM), EDTA (1 mM), reagents that react with cysteine; various detergents	pH: 4‐9 Temperature: 4°C‐37°C (max. at 34°C) Can accommodate most buffers	Sequence specificity more stringent than factor Xa, thrombin, and enterokinase Can be produced in E. coli
3Cb	Recombinant form of the 3C protease from human rhinovirus type 14	EVLFQ‐GP	PMSF, TLCK, leupeptin, zinc (100 mM), urea (1 M), guanidine (1 M)	pH: 3‐10 Temperature: 4°C‐37°C (max. at 4°C) Test activity in buffer of choice	High sequence specificity Can be produced in E. coli
Xac	Factor Xa is a serine protease that converts prothrombin to thrombin	I(E/N)GR‐ Will not cleave at site followed by P or R	PMSF, AEBSF, DFP, aprotinin, antithrombin III, antipain, α1‐antitypsin, chymostatin, hirudin, leupeptin, urea (100 mM), guanidine (10 mM), NaCl (100 mM), imidazole (100 mM)	pH: 6.5‐9 Temperature: 4°C‐37°C Activity is highly reduced in phosphate buffers compared to Tris or HEPES, CaCl2 should be included in cleavage reaction	Nonspecific proteolysis may occur at secondary sites Cleavage usually performed near physiological conditions
Thr	Thrombin is a serine protease that converts fibrinogen into fibrin	LVPR‐GS	PMSF, AEBSF, aprotinin, antithrombin III, antipain, α1‐antitypsin, chymostatin, hirudin, leupeptin, reducing agents	pH: 5‐10 Temperature: 4°C‐37°C (max. at 37°C) Can accommodate most buffers	Nonspecific proteolysis may occur at secondary sites Cleavage usually performed near physiological conditions
EntK	Catalytic subunit of bovine enterokinase	DDDDK‐	Serine protease inhibitors, PMSF, imidazole (250 mM), NaCl (250 mM), urea (2 M), SDS	pH: 6.0‐8.5 Temperature: 4°C‐37°C Activity is highly reduced in phosphate buffers compared to Tris or MES	Nonspecific proteolysis may occur at secondary sites

^aThe “–” indicates the site of cleavage within the single letter amino acid code.

^bSee Miyashita et al., 1992.

^cSee Nagai and Thogersen, 1984; Jenny et al., 2003.

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