After purifying a His-tagged recombinant protein, if you want to cleave off the tag, is TEV or thrombin more cost-effective?

The key depends on whether your fusion protein carries a canonical cleavage site. Thrombin recognizes LVPR/GS, often leaving a few residues, and may be affected by pH and salt; TEV recognizes ENLYFQ/G/S, with relatively fewer nonspecific cleavages. If your kinase contains internal stretches resembling basic amino acids, thrombin is more likely to cleave nonspecifically. It is recommended to first perform a 100 μg small-scale test: for thrombin, use 1–2 U/mg, cleave at 22°C for 2–4 h; for TEV, use 1:50–1:100, cleave at 4°C for 12–16 h. After cleavage, take 10 μL and run SDS-PAGE, comparing against an uncleaved control.

If the budget is tight and you still want to control nonspecific binding, you can have the lab express the His-TEV engineered mutant in-house, use Ni column crude extraction, add it directly to 2 mg of sample during cleavage, and then remove it with Ni. Although thrombin may look cheaper per unit, batch-to-batch variability, specific activity, and removal methods all increase labor costs for small-scale cleavage. Whichever you choose, it is recommended to reserve about 100–150 μg for pilot experiments and SDS-PAGE/activity assays; in the end, the actual amount of tag-free protein obtained may be only 1–1.5 mg.

I prefer to use His-tagged TEV or biotinylated thrombin first, then remove them by reverse Ni or affinity capture after cleavage to avoid residual exogenous protease interfering with subsequent enzyme activity and cell experiments. After tag cleavage, pass the sample through SEC once, or at least remove the His tag and His-TEV by Ni-NTA adsorption. For the buffer system, I recommend 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT or TCEP, and 5% glycerol. Do not use excessively long digestion times or excessive enzyme amounts; for 2 mg, first cleave a 0.2 mg small-scale sample to confirm the conditions before scaling up.

I have used both TEV and thrombin. In terms of cleavage efficiency, TEV can generally cleave fairly cleanly overnight at 4°C, and the ENLYFQ/G site also tolerates temperature and reducing agents relatively well; thrombin is faster, but when the unit amount is too high, nonspecific extra bands tend to appear. For a 2 mg small-scale prep, it is recommended to use TEV first, cleaving at 4°C at 1:50–1:100 (w/w) in a buffer containing 0.5–1 mM TCEP, sampling at time points and running gels, and stopping once the target band appears. In terms of cost, commercial TEV is not cheap, but if the lab has a His-TEV plasmid, it can be self-purified, which can reduce the cost per run.

Avoid nonspecific cleavage: Do not add too much thrombin; use 1–2 U/mg if possible, and do not incubate overnight at 37°C. TEV can be cleaved at 4°C; avoid inhibition with PMSF or similar. For kinase proteins, an activity assay must be performed after cleavage to confirm that no erroneous cleavage has occurred. Another practical approach is that if the His tag is at the N-terminus and far from the active pocket, the enzymatic activity of uncleaved and cleaved samples can first be compared; if the activity shows no significant change, skipping cleavage also reduces one step of loss. Do not expect to obtain high recovery in a single run; a 10–30% loss after cleavage is very common.