An overview of enzymatic reagents for the removal of affinity tags

Abstract

Although they are often exploited to facilitate the expression and purification of recombinant proteins, every affinity tag, whether large or small, has the potential to interfere with the structure and function of its fusion partner. For this reason, reliable methods for removing affinity tags are needed. Only enzymes have the requisite specificity to be generally useful reagents for this purpose. In this review, the advantages and disadvantages of some commonly used endo- and exoproteases are discussed in light of the latest information.

Fig. 1

Space-filling representations of the S1′ pockets of TEV protease (A) and TVMV protease (B). The canonical peptides that were co-crystallized with each enzyme are shown as stick representations. The side chains of the P1′ serine residues are encased in mesh. Note that the side chain of the P1′ serine projects along the surface of a shallow groove in TEV protease whereas the side chain of the corresponding serine points into a small, shallow pocket in TVMV protease.

Fig. 2

A generic strategy for protein purification that utilizes an affinity-tagged endoprotease. See text for discussion.

Fig. 3

Overcoming steric hindrance by inserting “spacer” residues between a protease recognition site and the N-terminus of the protein of interest. (A) Expression of MBP-SycH fusion protein with a TEV protease recognition site immediately adjacent to the N-terminal residue of SycH in the absence (−) or presence (+) of TEV protease. (B) Expression of an otherwise identical MBP-SycH fusion protein in which five consecutive glycine residues were inserted between the TEV protease recognition site and the N-terminal residue of SycH in the absence (−) and presence (+) of TEV protease.

Fig. 4

Relative processing efficiency (k_cat/K_M) of peptide substrates by MeCPA (black bars) and BoCPA (gray bars). The peptide substrates used were VSQNPKX, wherein X was the variable amino acid. No processing of peptides terminating in Arg, Lys, or Pro was observed for either enzyme. Data were compiled from .