TEV protease with dual affinity tags
Disclosed is using a TEV protease (wild type or mutant) suited for removal from a reaction mixture, wherein the TEV protease displays a dual affinity tag including a chitin binding domain (CBD) tag and a histidine tag, preferably where the CBD tag precedes the N-terminus of the protease and the CBD tag is preceded by the histidine tag, which is preferably a 6-mer histidine tag; and optionally further including linkers, preferably Gly-Ser linkers, and more preferably a 6-mer Gly-Ser linker, between the tags. A linker, also preferably a Gly-Ser linker, can also follow the CBD tag and precede the protease portion. In certain embodiments, no such linkers are present and in other embodiments, only one such linker is present.
1 . A process of cleaving a target protein with a fusion protein having sequences of Tobacco Etch Virus protease, wild type or mutant, with two affinity tags, and then removing the fusion protein after cleavage by binding to either or both of the affinity tags, comprising:
generating the fusion protein where a chitin binding domain precedes the residues representing the N-terminus of the protease, and the chitin binding domain is preceded by a poly-histidine tag;
combining the fusion protein with a substrate for cleavage by the fusion protein in a reaction solution under reaction conditions; and
binding the fusion protein to a solid support having chitin on its support surface or wherein the solid support bears a metal.
2 . The process of claim 1 wherein the solid support is a resin or a magnetic bead.
3 . The process of claim 2 wherein the magnetic bead includes nickel.
4 . The process of claim 2 wherein the solid support is a magnetic bead and further including removing the magnetic beads bearing the bound fusion protein from the reaction solution using magnetic attraction.
5 . The process of claim 1 wherein the fusion protein has a series of glycine and serine residues between the poly-histidine tag and the chitin binding domain.
6 . The process of claim 5 further including a series of glycine and serine residues between the chitin binding domain and the residues at the N-terminus of the protease.
7 . The process of claim 6 wherein the linkers are both 6-mers.
8 . The process of claim 6 wherein the linkers are both: GSGSSG (SEQ ID NO: 7).
9 . The process of claim 1 wherein the fusion protein includes a MKI leader sequence.
10 . A process of cleaving a target protein with a fusion protein having the sequence of SEQ ID NO:2 with two affinity tags, and then removing the fusion protein after cleavage by binding to either or both of the affinity tags, comprising:
combining the fusion protein with a substrate for cleavage by the fusion protein in a reaction solution under reaction conditions; and
binding the fusion protein to a solid support having chitin on its support surface or wherein the solid support bears a metal.
11 . The process of claim 10 wherein the solid support is a resin or a magnetic bead.
12 . The process of claim 11 wherein the magnetic bead includes nickel.
13 . The process of claim 11 wherein the solid support is a magnetic bead and further including removing the magnetic beads bearing the bound fusion protein from the reaction solution using magnetic attraction.