Which of the following proteases will give a clean cut at the N-terminus (i.e. have no additional amino acids present once treated with the protease)?

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To determine which proteases yield a truly seamless N-terminus after cleavage, we must examine the reported cleavage recognition sequences and what residues remain at the new N-terminus post-cleavage.
Option a. TEV: TEV protease recognizes ENLYFQ/G and cleaves between Q and G. This means a Gly (G) residue is left just after the cleavage site, becoming the new N-terminus of the downstream sequence. Therefore, TEV does not provide a completely clean N-terminus with no extra residues. This makes option a incorrect as a universal clean-cut protease for the N-terminus.
Option b. EntK: EntK (Enterokinase) recognizes the DDDDK sequence and cleaves after the lysine (K). The canonical site is DDDDK|X, where X is the next amino acid of the target. Since the cut occurs immediately before X, there are no extra residues appended to the N-terminus of the downstream protein beyond the intended sequence X. This yields a clean N-terminus, making option b a correct choice.
Option c. Xa: Factor Xa recognizes the IEGR sequence and cleaves after R (IEGR|). Similar to EntK, the cut occurs right before the downstream residue, so the N-terminus starts with the intended residue X without additional residues. Hence, Xa yields a clean N-terminus, making option c a correct choice.
Option d. Thr: This option refers to thrombin, which recognizes LVPR|GS and cleaves after R. Cleavage leaves a Gly-Ser (GS) dipeptide at the new N-terminus in many designs, introducing extra residues. Thus thrombin does not provide a completely clean N-terminus. This makes option d incorrect.
Option e. PreScission: PreScission protease recognizes the LEVLFQ/GP site and cleaves between Q and G, often leaving a GP dipeptide at the N-terminus of the downstream product. This results in additional residues at the new N-terminus, so it is not a clean cut. Therefore option e is incorrect.
In summary, the proteases EntK and Xa (Options b and c) are the ones that can give a truly clean N-terminus after cleavage, whereas TEV, Thrombin, and PreScission generally leave extra residues at the N-terminus.

BPS3021 S1 2025 CYL | Protein purification & characterisation

Which of the following proteases will give a clean cut at the N-terminus (i.e. have no additional amino acids present once treated with the protease)?

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