Tag removal from proteins expressed in pro- and eukaryotic hosts

Patent metadata

Field	Value
Publication number	US-10378003-B2
Application number	US-201515525487-A
Country	US
Kind code	B2
Filing date	Nov 10, 2015
Priority date	Nov 10, 2014
Publication date	Aug 13, 2019
Grant date	Aug 13, 2019

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.

First claim

The invention claimed is:

1. 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 (His 14 -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 MgCl 2 , 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 (His 14 -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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

2. The protease of claim 1 , wherein the protease comprises the amino acid sequence of amino acids 14-384 of SEQ ID NO: 1 (xlAtg4B).

3. The protease of claim 1 , wherein the protease is capable of cleaving
(i) at least 90% of a 100-fold, preferably 150-fold, more preferably 200-fold molar excess of a native substrate protein shown in SEQ ID NO: 3 (His 14 -xlLC3B-MBP); and/or
(ii) at least 90% of a 150-fold, preferably 200-fold, more preferably 300-fold molar excess of a native substrate protein shown in SEQ ID NO: 5 (His 14 -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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

4. The protease of claim 1 , wherein the protease is capable of cleaving
(i) at least 90% of a 500-fold, preferably 1000-fold, more preferably 1500-fold, most preferably 2000-fold molar excess of a native substrate protein shown in SEQ ID NO: 3 (His 14 -xlLC3B-MBP); and/or
(ii) at least 90% of a 2000-fold, preferably 3000-fold, more preferably 4000-fold, even more preferably 5000-fold, still more preferably 6000-fold, most preferably 6600-fold molar excess of a native substrate protein shown in SEQ ID NO: 5 (His 14 -xlGATE16-MBP);

at conditions of 1 hour incubation at 25° 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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

5. The protease of claim 1 , wherein the protease is capable of cleaving at least 90% of a 100-fold molar excess of native substrate protein variants in which only residue 152 in SEQ ID NO: 3 (the P 1 ′ position of His 14 -xlLC3B-MBP) has been mutated to Met, Tyr, Arg or Glu relative to SEQ ID NO: 3 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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

6. The protease of claim 1 , wherein the protease is capable of cleaving at least 50% of a 200-fold molar excess of a native substrate protein as shown in SEQ ID NO: 3 (His 14 -xlLC3B-MBP) within one hour at 0° C. at high-salt conditions of 100 μM initial concentration of substrate protein in a buffer consisting of 1.5 M NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl 2 , 250 mM sucrose, 2 mM DTT.

7. The protease of claim 1 , wherein the protease cleaves at stringent conditions any of the substrates shown in SEQ ID NO: 6 (His 10 -ZZ-TEV-MBP), SEQ ID NO: 7 (His 14 -bdNEDD8-MBP), SEQ ID NO: 8 (His 14 -bdSUMO-MBP), SEQ ID NO: 9 (His 14 -xlUb-MBP), or SEQ ID NO: 22 (His 14 -SUMOstar-MBP) at least 10 000 fold less efficiently than the substrate shown in SEQ ID NO: 3 (His 14 -xlLC3B-MBP), wherein stringent conditions are defined as 3 hour incubation at 25° C., 20 μM protease, 100 μM initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl 2 , 250 mM sucrose, 2 mM DTT.

8. The protease of claim 1 , wherein the protease, if the protease does not comprise a polyHis-tag, is capable of cleaving a substrate protein as shown in SEQ ID NO: 25 (His 14 -IF2d1-xlLC3B-MBP) immobilized on a Ni(II) chelate resin with at least 10% efficiency as compared to the non-immobilised substrate 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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

9. The protease of claim 1 , wherein the protease retains at least 50% of its activity when pre-incubated for 16 h at 42° C. in the absence of oxygen in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl 2 , 250 mM sucrose, 20 mM DTT, as compared to said non-treated protease, if tested using a native substrate protein shown in SEQ ID NO: 3 (His 14 -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 MgCl 2 , 250 mM sucrose, 2 mM DTT.

10. The protease of claim 1 , wherein the protease further comprises a poly-His tag, a MBP-tag or a ZZ-tag.

11. The protease of claim 1 , wherein the protease further comprises an affinity tag.

12. A protease consisting of the amino acid sequence of amino acids 14-384 of SEQ ID NO: 1 (xlAtg4B).

13. A method of removing a protein tag, which comprises contacting said protein tag the protease according to claim 1 .

14. The method of claim 13 , wherein the protease is used for on-column cleavage in an affinity chromatographic purification step.

Assignees

• Max Planck Gesellschaft

Inventors

• Frey Steffen
• Goerlich Dirk

Classifications

• C12N9/641 Primary

  Cysteine endopeptidases (3.4.22) · CPC title

• C12N9/60 Primary

  from yeast · CPC title

• C12N9/6405

  not being snakes · CPC title

• C07K1/22

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title