Protease specific for a novel sumo-derived protease cleavage site

A variant protease,

• wherein said variant protease has at least 96% sequence identity over the full-length of the polypeptide of SEQ ID NO:6,
• wherein said variant protease comprises an amino acid substitution at the position corresponding to position 33 of the protein of SEQ ID NO:
• 6, wherein the amino acid at said position is substituted by an amino acid selected from the group consisting of S, H, Q, A, G, and C, and
• wherein said variant protease cleaves a first protease cleavage site (PCS) in a first fusion protein that comprises a maltose-binding protein (MBP) and said first PCS, wherein said first PCS is fused to the N-terminus of the MBP (first PCS-MBP fusion protein), wherein said first PCS has the amino acid sequence of SEQ ID NO:
• 41 and the MBP has the amino acid sequence of SEQ ID NO:
• 71, more efficiently after the C-terminal Gly-Gly motif of the first PCS of SEQ ID NO:
• 41 than said variant protease cleaves
• (i) a second PCS in a second fusion protein that comprises a MBP and said second PCS, wherein said second PCS is fused to the N-terminus of the MBP (second PCS-MBP fusion protein), wherein said second PCS has the amino acid sequence of SEQ ID NO:
• 4 and the MBP has the amino acid sequence of SEQ ID NO:
• 71, wherein the cleavage occurs after the C-terminal Gly-Gly motif of the second PCS of SEQ ID NO:
• 4, or
• (ii) a third PCS in a third fusion protein that comprises a MBP and said third PCS, wherein said third PCS is fused to the N-terminus of the MBP (third PCS-MBP fusion protein), wherein said third PCS has the amino acid sequence of SEQ ID NO:
• 3 and the MBP has the amino acid sequence of SEQ ID NO:
• 71, wherein the cleavage occurs after the C-terminal Gly-Gly motif of the third PCS of SEQ ID NO:
• 3,
• when tested in a buffer under standard conditions of 1 hour incubation at 21° C., wherein said buffer comprises an initial concentration of the first, second and third PCS-MBP fusion proteins of 100 μM, and wherein the buffer comprises a pH of 7.5, Tris/HCl at a concentration of 45 mM, NaCl at a concentration of 250 mM, MgCl2 at a concentration of 2 mM, sucrose at a concentration of 250 mM, and DTT at a concentration of 10 mM.