Tag removal from proteins expressed in pro- and eukaryotic hosts
AssigneeMax-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
InventorsSteffen Frey, Dirk Goerlich
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Abstract
The present invention belongs to the field of biotechnology. More specifically, the present invention provides a protease, a non-naturally occurring fusion protein comprising a corresponding protease recognition site, expression vectors encoding same, host cells comprising said expression vectors, kit of parts as well as methods applying the protease, fusion protein, and uses thereof, as defined in the claims. The presently disclosed protease/protease recognition site is particularly useful in methods requiring an orthogonal set of proteases, and is suitable for use in both prokaryotic and selected eukaryotic expression systems.
Claims14
A protease comprising an amino acid sequence with at least 97% identity over amino acids 14 384 of SEQ ID NO:
- 1 (xlAtg4B), with the proviso that the protease is not the protease of SEQ ID NO:
- 1,
- wherein said protease is capable of cleaving the protease recognition site (PRS) according to SEQ ID NO:
- 2 (xlLC3B) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO:
- 1, if tested using a native substrate protein shown in SEQ ID NO:
- 3 (His14-xlLC3B-MBP) and 500 nM of said protease at standard conditions of 1 hour incubation at 0° C., 100 μM initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl2, 250 mM sucrose, 2 mM DTT and/or
- wherein said protease is capable of cleaving the protease recognition site (PRS) according to SEQ ID NO:
- 4 (xlGATE16) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO:
- 1, if tested using 500 nM of said protease and a native substrate protein shown in SEQ ID NO:
- 5 (His14-xlGATE16-MBP) at standard conditions of 1 hour incubation at 0° C., 100 μM initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl2, 250 mM sucrose, 2 mM DTT.
A protease consisting of the amino acid sequence of amino acids 14-384 of SEQ ID NO:
- 1 (xlAtg4B).
Dependencies are inferred from the claim text.