TAG REMOVAL FROM PROTEINS EXPRESSED IN PRO- AND EUKARYOTIC HOSTS
20180195056 ยท 2018-07-12
Assignee
Inventors
Cpc classification
C07K1/22
CHEMISTRY; METALLURGY
International classification
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
Claims
1. A protease comprising an amino acid sequence with at least 80% identity over amino acids 25-384 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.sub.14-xlLC3B-MBP) and 500 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.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.sub.14-xlGATE16-MBP) at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
2. The protease of claim 1, wherein the protease comprises an amino acid sequence with at least 80% identity to amino acids 14-384 of SEQ ID NO: 1 (xlAtg4B).
3. The protease of claim 1 or 2, wherein the protease comprises an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 1 (xlAtg4B).
4. The protease of any one of claims 1-3, wherein the protease comprises the amino acid sequence of amino acids 25-384 of SEQ ID NO: 1 (xlAtg4B).
5. The protease of any one of claims 1-4, wherein the protease comprises the amino acid sequence of amino acids 14-384 of SEQ ID NO: 1 (xlAtg4B).
6. The protease of any one of claims 1-5, wherein the protease consists of the amino acid sequence of amino acids 14-384 of SEQ ID NO: 1 (xlAtg4B).
7. The protease of any one of claims 1-5, wherein the protease consists of the amino acid sequence of amino acids 25-384 of SEQ ID NO: 1 (xlAtg4B).
8. The protease of any one of claims 1-7, 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.sub.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.sub.14-xlGATE16-MBP); at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
9. The protease of any one of claims 1-8, 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.sub.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.sub.i4-xlGATE16-MBP); at conditions of 1 hour incubation at 25 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
10. The protease of any one of claims 1-9, 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.sub.1 position of His.sub.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 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
11. The protease of any one of claims 1-10, 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.sub.14-xlLC3B-MBP) within one hour at 0 C. at high-salt conditions of 100 M initial concentration of substrate protein in a buffer consisting of 1.5 M NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
12. The protease of any one of claims 1-11, wherein the protease cleaves at stringent conditions any of the substrates shown in SEQ ID NO: 6 (His.sub.10-ZZ-TEV-MBP), SEQ ID NO: 7 (His.sub.14-bdNEDD8-MBP), SEQ ID NO: 8 (His.sub.14-bdSUMO-MBP), SEQ ID NO: 9 (His.sub.14-xlUb-MBP), or SEQ ID NO: 22 (His.sub.14-SUMOstar-MBP) at least 10 000 fold less efficiently than the substrate shown in SEQ ID NO: 3 (His.sub.14-xlLC3B-MBP), wherein stringent conditions are defined as 3 hour incubation at 25 C., 20 M protease, 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
13. The protease of any one of claims 1-12, 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.sub.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 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
14. The protease of any one of claims 1-13, 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.sub.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.sub.14-xlLC3B-MBP) and 500 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
15. The protease of any one of claims 1-14, wherein the protease further comprises an affinity tag, preferably a poly-His tag, a MBP-tag or a ZZ-tag.
16. A non-naturally occurring fusion protein, comprising a protease recognition site (PRS), which PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 2 (xlLC3B); or (ii) a derivative of (i) with an amino acid sequence having at least 75% identity over the full length of SEQ ID NO: 2 (xlLC3B), wherein the protease shown in SEQ ID NO: 1 (xlAtg4B) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the amino acid sequence of SEQ ID NO: 2 (xlLC3B), under identical conditions of 1 hour incubation at 0 C., 500 nM protease, 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
17. A non-naturally occurring fusion protein, comprising a protease recognition site (PRS), which PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 4 (xlGATE16); or (ii) a derivative of (i) with an amino acid sequence having at least 75% identity over the full length of SEQ ID NO: 4 (xlGATE16), wherein the protease shown in SEQ ID NO: 1 (xlAtg4B) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the amino acid sequence of SEQ ID NO: 4 (xlGATE16), under identical conditions of 1 hour incubation at 0 C., 200 nM protease, 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
18. The fusion protein of claim 16 or 17, further comprising an affinity tag, wherein the affinity tag is located in the fusion so that it is cleaved off if the fusion protein is incubated with the protease shown in SEQ ID NO: 1 (xlAtg4B), preferably wherein the affinity tag is N-terminal from the PRS.
19. An expression vector, comprising a multiple cloning site in functional linkage with a nucleic acid sequence encoding the protease recognition site as defined in claim 16 or 17.
20. The expression vector of claim 19, wherein the nucleic acid sequence further encodes an affinity tag as defined in claim 18.
21. An expression vector, comprising a nucleic acid sequence encoding the fusion protein according to any one of claims 16-18.
22. A host cell, comprising an expression vector according to any one of claims 19-21.
23. The host cell of claim 22, wherein the host cell is a eukaryotic host cell, wherein the eukaryotic cell is a fungal cell, or a plant cell.
24. The host cell of claim 23, wherein the host cell is a fungal cell, preferably a yeast cell, more preferably wherein the cell is of the genus Saccharomyces, even more preferably wherein the host cell is a cell of Saccharomyces cerevisiae.
25. The host cell of claim 23, wherein the host cell is a plant cell, preferably wherein said plant cell is a cell of the order Poales, more preferably wherein said cell is of the family Poaceae, even more preferably wherein said cell is of the subfamily Pooideae, still more preferably wherein said cell is of the tribe Triticeae, and most preferably, wherein said cell is of the genus Triticum.
26. The host cell of claim 22, wherein the host cell is a cell of E. coli.
27. The host cell of any one of claims 22-26, wherein the host cell further expresses a protease as defined in any one of claims 1-15.
28. Use of the protease according to any one of claims 1-15 for removing a protein tag, preferably wherein the protein tag is an affinity tag.
29. The use of claim 28, wherein the protease is used for on-column cleavage in an affinity chromatographic purification step.
30. Use of an expression vector according to claims 19-21 or of a host cell according to any one of claims 22-26 in the production of a fusion protein in a eukaryotic expression system.
31. A method for purifying a stoichiometric protein complex composed of at least two subunits from a mixture, said mixture comprising said protein complex and monomers of said at least two subunits, wherein said at least two subunits comprised in said mixture each comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), wherein the ATs of each of said at least two subunits differ from each other and allow affinity chromatography being selective for each AT, and wherein the PRS of each of said at least two subunits is cleavable by a protease, which protease is orthogonal to the PRS of the other subunit(s), wherein the method comprises the steps of a) subjecting the mixture to a first affinity chromatography selective for the AT of the first of said at least two subunits, whereby (i) the protein complex binds to the affinity resin via the AT of the first subunit, and (ii) impurities are washed off the column, and (iii) the protein complex is eluted from the column and the AT of the first subunit is cleaved off, or the protein complex is eluted by on-column cleavage, using said orthogonal protease which is specific for the PRS of said first subunit, and (iv) optionally removing the cleaved off AT of the first subunit; and b) subjecting the eluate from step a) to a second affinity chromatography selective for the AT of the second of said at least two subunits, whereby (i) the protein complex binds to the affinity resin via the AT of the second subunit, and (ii) impurities are washed off the column, and (iii) the protein complex is eluted from the column and the AT of the second subunit is cleaved off, or the protein complex is eluted by on-column cleavage, using said orthogonal protease which is specific for the PRS of said second subunit, and (iv) optionally removing the cleaved off AT of the second subunit; characterized in that one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 2 (xlLC3B); or (ii) a derivative of (i) with an amino acid sequence having at least 75% identity over the full length of SEQ ID NO: 2 (xlLC3B), wherein the protease shown in SEQ ID NO: 1 (xlAtg4B) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the amino acid sequence of SEQ ID NO: 2 (xlLC3B), under identical conditions of 500 nM protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; or (i) an amino acid sequence as shown in SEQ ID NO: 4 (xlGATE16); or (ii) a derivative of (i) with an amino acid sequence having at least 75% identity over the full length of SEQ ID NO: 4 (xlGATE16), wherein the protease shown in SEQ ID NO: 1 (xlAtg4B) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the amino acid sequence of SEQ ID NO: 4 (xlGATE16), under identical conditions of 200 nM protease, 1 hour incubation at 0 C., 100 M io initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; and wherein the AT of the subunit comprising said PRS is cleaved off using a protease according to any one of claims 1-15.
32. The method of claim 31, wherein one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 2 (xlLC3B); or (ii) a derivative of (i) with an amino acid sequence having at least 75% identity over the full length of SEQ ID NO: 2 (xlLC3B), wherein the protease shown in SEQ ID NO: 1 (xlAtg4B) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the amino acid sequence of SEQ ID NO: 2 (xlLC3B), under identical conditions of 500 nM protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein one PRS comprises, more preferably consists of an amino acid sequence as shown in SEQ ID NO: 2 (xlLC3B).
33. The method of claim 31 or 32, wherein in step a) (iii) and/or step b) (iii) the protein complex is eluted by on-column cleavage.
34. The method of any one of claims 31-33, wherein the method further comprises the step of c) removing the protease from the eluate originating from the last affinity chromatography.
35. The method of any one of claims 31-34, wherein one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 9 (bdSUMO); or (ii) a PRS derivative of (i) with an amino acid sequence having at least 60% identity over the full length of SEQ ID NO: 10 (bdSUMO), wherein the protease shown in SEQ ID NO: 11 (bdSENP1.sup.248-481) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 10, under identical conditions of 30 nM protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; and wherein the AT of the subunit comprising said PRS is cleaved off using (i) a protease comprising, preferably consisting of the amino acid sequence shown in amino acids 1-224 of SEQ ID NO: 11 (bdSENP1.sup.248-481), or (ii) a protease derivative of (i) having an amino acid sequence with at least 45% identity over the full length of SEQ ID NO: 11, wherein said protease derivative is capable of cleaving the PRS according to ID NO: 10 (bdSUMO) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 8 (His.sub.ia-bdSUMO-MBP) and 30 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the subunit is eluted from the column using (i) the protease shown in in amino acids 1-224 of SEQ ID NO: 11 (bdSENP1.sup.248-481).
36. The method of any one of claims 31-35, wherein one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 12 (bdNEDD8); or (ii) a PRS derivative of (i) with an amino acid sequence having at least 85% identity over the full length of SEQ ID NO: 12, wherein the protease shown in SEQ ID NO: 13 (bdNEDP1) is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 12 under identical conditions of 300 nM protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; and wherein the AT of the subunit comprising said PRS is cleaved off using (i) a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 13 (bdNEDP1), or (ii) a protease derivative of (i) having an amino acid sequence with at least 35% identity over the full length of SEQ ID NO: 13 (bdNEDP1), wherein said protease derivative, cleaves the PRS according to SEQ ID NO: 12 (bdNEDD8) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 7 (His.sub.14-bdNEDD8-MBP) and 300 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the subunit is eluted from the column using the protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 13 (bdNEDP1).
37. The method of any one of claims 31-36, wherein at least one PRS comprises, preferably consists of the TEV protease recognition site shown in SEQ ID NO: 14; and wherein the AT of the subunit comprising said PRS is cleaved off using (i) a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 15 or 16 (TEV protease), or (ii) a protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 15 or 16, wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 14 (TEV) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 6 (His.sub.to-ZZ-TEV-MBP) and 10 M of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
38. The method of any one of claims 31-37, wherein at least one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 17 (xlUb); or (ii) a PRS derivative of (i) with an amino acid sequence having at least 80% identity over the full length of SEQ ID NO: 17, wherein the protease shown in SEQ ID NO: 18 (xlUsp2), is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 17 under identical conditions of 1 M protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; and wherein the AT of the subunit comprising said PRS is cleaved off using (i) a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 18 (xlUsp2), or (ii) a protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 18, wherein said protease derivative is capable of cleaving the PRS according to ID NO: 17 (xlUb) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 9 (His.sub.14-xlUb-MBP) and 1 M of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
39. The method of any one of claims 31-38, wherein at least one PRS comprises, preferably consists of (i) an amino acid sequence as shown in SEQ ID NO: 23 (SUMOstar); or (ii) a PRS derivative of (i) with an amino acid sequence having at least 80% identity over the full length of SEQ ID NO: 23, wherein the protease shown in SEQ ID NO: 24 (SUMOstar protease), is capable of cleaving said PRS derivative with at least 20% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 23 under identical conditions of 30 nM protease, 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; and wherein the AT of the subunit comprising said PRS is cleaved off using (i) a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 24 (SUMOstar protease), or (ii) a protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 24, wherein said protease derivative is capable of cleaving the PRS according to ID NO: 23 (SUMOstar) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 22 (His.sub.14-SUMOstar-MBP) and 30 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT.
40. The method of any one of claims 31-39, wherein the protein complex is composed of 2 different subunits, preferably with a stoichiometry of 1:1; or wherein the protein complex is composed of 3 different subunits, preferably with a stoichiometry of 1:1:1; or wherein the protein complex is composed of 4 different subunits, preferably with a stoichiometry of 1:1:1:1.
41. The method of any one of claims 31-40, wherein the mixture originates from a suitable fungal expression host cell, preferably wherein the host cell is a yeast cell, more preferably wherein the cell is of the genus Saccharomyces, most preferably wherein the host cell is a cell of Saccharomyces cerevisiae.
42. The method of claim 41, wherein one subunit comprises the PRS xlLC3B or a PRS derivative thereof as defined in claim 31, and wherein the elution is carried out using the protease as defined in claim 31; and wherein the other subunit comprises the PRS bdNEDD8 or a PRS derivative thereof as defined in claim 36 and wherein the elution is carried out using the protease as defined in claim 36.
43. The method of claim 41, wherein one subunit comprises the PRS xlLC3B or a PRS derivative thereof as defined in claim 31, and wherein the elution is carried out using the protease as defined in claim 31; and wherein the other subunit comprises the PRS SUMOstar or a PRS derivative thereof as defined in claim 39 and wherein the elution is carried out using the protease as defined in claim 39.
44. The method of claim 41, wherein one subunit comprises the PRS xlLC3B or a PRS derivative thereof as defined in claim 31, and wherein the elution is carried out using the protease as defined in claim 31; wherein a second subunit comprises the PRS bdNEDD8 or a PRS derivative thereof as defined in claim 36 and wherein the elution is carried out using the protease as defined in claim 36; and wherein a third subunit comprises the PRS SUMOstar or a PRS derivative thereof as defined in claim 39 and wherein the elution is carried out using the protease as defined in claim 39.
45. The method of any one of claims 31-40, wherein the mixture originates from a suitable plant expression host cell, preferably wherein said plant cell is a cell of the order Poales, more preferably wherein said cell is of the family Poaceae, even more preferably wherein said cell is of the subfamily Pooideae, still more preferably wherein said cell is of the tribe Triticeae, and most preferably, wherein said cell is of the genus Triticum.
46. The method of claim 45, wherein one subunit comprises the PRS or a PRS derivative thereof as defined in claim 31 or 32, and wherein the elution is carried out using the protease as defined in claim 31; and wherein the other subunit comprises the PRS SUMOstar or a PRS derivative thereof as defined in claim 39 and wherein the elution is carried out using the protease as defined in claim 39.
47. The method of any one of claims 31-46, wherein the one subunit comprises a poly-His tag, and preferably wherein the other subunit comprises a ZZ tag.
48. The method of any one of claims 31-47, wherein the mixture originates from a single lysate or supernatant or a pre-purified solution comprising all subunits of the protein complex.
49. The method of any one of claims 31-48, wherein the mixture originates from a mixture of lysates and/or supernatants and/or pre-purified solutions.
50. The method of any one of claims 31-49, wherein step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
51. The method of any one of claims 31-50, wherein the protease from the eluate originating from the last affinity chromatography prior to step c) comprises an affinity tag, preferably a poly-His tag or a ZZ tag, and wherein step c) is an affinity chromatography step, whereby the protease binds to the affinity resin, and the protein complex is collected in the flow-through.
52. The method of any one of claims 31-51, wherein the subunit(s) further comprise a spacer between the AT and the PRS, and/or between the PRS and the subunit; preferably wherein the subunit(s) further comprise a spacer between the AT and the PRS.
53. A kit of parts, comprising (i) the protease according to any one of claims 1-15, and (ii) an expression vector according to any one of claims 19-21 or a host cell according to any one of claims 22-27.
54. A kit of parts, comprising (i) the protease according to any one of claims 1-15, and at least one protease selected from the group of proteases consisting of (ii) a protease having an amino acid sequence with at least 45% identity over the full length of SEQ ID NO: 11 (bdSENP1), wherein said protease is capable of cleaving the PRS according to ID NO: 10 (bdSUMO) with at least 20% activity as compared to the parent protease of SEQ ID NO: 10 (bdSENP1), if tested using a native substrate protein shown in SEQ ID NO: 8 (His.sub.u-bdSUMO-MBP) and 30 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the protease comprises the amino acid sequence shown as amino acids 1-224 in SEQ ID NO: 11 (bdSENP1.sup.248-481); more preferably wherein the protease consists of the amino acid sequence shown as amino acids 1-224 in SEQ ID NO: 11 (bdSENP1.sup.248-481); (iii) a protease having an amino acid sequence with at least 35% identity over the full length of SEQ ID NO: 13 (bdNEDP1), wherein said protease cleaves the PRS according to SEQ ID NO: 12 (bdNEDD8) with at least 20% activity as compared to the parent protease of SEQ ID NO: 13 (bdNEDP1), if tested using a native substrate protein shown in SEQ ID NO: 7 (His.sub.14-bdNEDD8-MBP) and 300 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 13 (bdNEDP1); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 13 (bdNEDP1); (iv) a protease having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 15 or 16, wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 14 (TEV) with at least 20% activity as compared to the parent protease of SEQ ID NO: 15 or 16, if tested using a native substrate protein shown in SEQ ID NO: 6 (His.sub.10-ZZ-TEV-MBP) and 10 M of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 15 or 16, more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 15 or 16; (v) a protease having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 18 (xlUsp2), wherein said protease is capable of cleaving the PRS according to ID NO: 17 (xlUb) with at least 20% activity as compared to the parent protease of SEQ ID NO: 18 (xlUsp2), if tested using a native substrate protein shown in SEQ ID NO: 9 (His.sub.14-xlUb-MBP) and 1 M of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT. preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 18 (xlUsp2); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 18 (xlUsp2); (vi) a protease having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 24 (SUMOstar protease), wherein said protease is capable of cleaving the PRS according to ID NO: 23 (SUMOstar) with at least 20% activity as compared to the parent protease as defined in (i), if tested using a native substrate protein shown in SEQ ID NO: 22 (His.sub.14-SUMOstar-MBP) and 30 nM of said protease at standard conditions of 1 hour incubation at 0 C., 100 M initial concentration of substrate protein in a buffer consisting of 250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 24 (SUMOstar protease); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 24 (SUMOstar protease).
55. The kit of parts of claim 54, wherein at least one protease further comprises an affinity tag, preferably a poly-His tag or a ZZ tag.
56. The kit of parts of claim 54 or 55, which comprises the proteases as defined in (i) and (ii).
57. The kit of parts of claim 54 or 55, which comprises the proteases as defined in (i) and (iii).
58. The kit of parts of claim 54 or 55, which comprises the proteases as defined in (i) and (vi).
59. Use of a kit of parts as defined in any one of claims 54-58 in a method of purifying stoichiometric protein complexes comprising at least two subunits, preferably wherein the method is further defined as in any one of claims 31-52.
60. Use of a kit of parts as defined in any one of claims 54-58 for on-column cleavage in an affinity chromatography.
61. Use of a kit of parts as defined in any one of claims 56-58 in a method of purifying stoichiometric protein complexes comprising at least two subunits from a mixture originating from a eukaryotic expression host cell, preferably wherein said eukaryotic cell is a fungal cell or plant cell.
62. The use of claim 61, wherein the host cell is a fungal cell, preferably wherein the host cell is a yeast cell, more preferably wherein the cell is of the genus Saccharomyces, most preferably wherein the host cell is a cell of Saccharomyces cerevisiae.
63. The use of claim 61, wherein the host cell is a plant cell, preferably wherein said plant cell is a cell of the order Poales, more preferably wherein said cell is of the family Poaceae, even more preferably wherein said cell is of the subfamily Pooideae, still more preferably wherein said cell is of the tribe Triticeae, and most preferably, wherein said cell is of the genus Triticum.
Description
DESCRIPTION OF THE FIGURES
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[0196]
[0197]
[0198]
[0199]
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[0201]
[0202]
[0203]
[0204]
[0205]
[0206]
DESCRIPTION OF THE SEQUENCES
[0207]
TABLE-US-00002 (XenopuslaevisAtg4B(xlAtg4B)) SEQIDNO:1 MDAATLTYDTLRFADTPDFPETAEPVWVLGRKYSALTEKEQLLNDITSRL WFTYRRNFQAIGGTGPTSDTGWGCMLRCGQMIFAQALICRHVGRDWRWDK QKPKGEYLNILTAFLDKKDSYYSIHQIAQMGVGEGKYIGQWYGPNTVAQV LRKLAVFDQWSSIAVHIAMDNTVVVDEIRRLCRAGSGESSDAGALSNGYT GDSDPSCAQWKPLVLLIPLRLGLSEINEAYIETLKHCFMVPQSLGVIGGR PNSAHYFIGYVGDELIYLDPHTTQLSVEPSDCSFIEDESFHCQHPPCRMH VSEIDPSIAVGFFCSSQEDFEDWCQHIKKLSLSGGALPMFEVVDQLPLHL SNPDVLNLTPDSSDADRLDRFFDSEDEEFEILSL (XenopuslaevisLC3B(xlLC3B)) SEQIDNO:2 MPSEKTFKQRRSLEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKT KFLVPDHVNMSELIKIIRRRLQLNSNQAFFLLVNGHSMVSVSTPISEVYE REKDEDGFLYMVYASQETFG (His.sub.14-xlLC3B-MBP) SEQIDNO:3 MSKHHHHSGHHHTGHHHHSGSHHHTGGSSGSESSEKTFKQRRSLEQRVED VRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRR RLQLNSNQAFFLLVNGHSMVSVSTPISEVYEREKDEDGFLYMVYASQETF GAGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSA LMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTV LPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKD KPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTNGTGC (XenopuslaevisGATE16(xlGATE16)) SEQIDNO:4 MKWMFKEDHSLEHRCVESAKIRAKYPDRVPVIVEKVSGSQIVDIDKRKYL VPSDITVAQFMWIIRKRIQLPSEKAIFLFVDKTVPQSSLTMGQLYEKEKD EDGFLYVAYSGENTFG (His.sub.14-xlGATE16-MBP) SEQIDNO:5 MSKHHHHSGHHHTGHHHHSGSHHHTGGSSGSESSMKWMFKEDHSLEHRCV ESAKIRAKYPDRVPVIVEKVSGSQIVDIDKRKYLVPSDITVAQFMWIIRK RIQLPSEKAIFLFVDKTVPQSSLTMGQLYEKEKDEDGFLYVAYSGENTFG AGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVIVEHPDKLEEKF PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAV RYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSAL MFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVD LIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDK PLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAV INAASGRQTVDEALKDAQTNGTGC (His.sub.10-ZZ-TEV-MBP) SEQIDNO:6 MHHHHHHHHHHGSNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPS QSANLLAEAKKLNDAQAPKVAMNKFNKEQQNAFYEILHLPNLNEEQRNAF IQSLKDDPSQSANLLAEAKKLNDAQAPKVAMSGENLYFQGTKTEEGKLVI WINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI IFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIA VEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTD YSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFV GVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEE ELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTNGTGC (His.sub.14-bdNEDD8-MBP) SEQIDNO:7 MSKHHHHSGHHHTGHHHHSGSHHHSGTMIKVKTLTGKEIEIDIEPTDTID RIKERVEEKEGIPPVQQRLIYAGKQLADDKTAKDYNIEGGSVLHLVLALR GGAGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTVVEEIPALDKELKAKGK SALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTF LVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVN KDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVR TAVINAASGRQTVDEALKDAQTNGTGC (His.sub.14-bdSUMO-MBP) SEQIDNO:8 MSKHHHHSGHHHTGHHHHSGSHHHSGSAAGGEEDKKPAGGEGGGAHINLK VKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMTAIAFLFDGRRLRAEQ TPDELEMEDGDEIDAMLHQTGGAGTKTEEGKLVIWINGDKGYNGLAEVGK KFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGL LAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGK YDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMT INGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQ KGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNGTGC (His.sub.14-xlUb-MBP) SEQIDNO:9 MSKHHHHSGHHHTGHHHHSGSHHHTGGSSGSESSMQIFVKTLTGKTITLE VEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKEST LHLVLRLRGGAGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTE HPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDK LYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKE LKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGA KAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTS KVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE GLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTNGTGC (bdSUMOaminoacids21-97) SEQIDNO:10 HINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMTAIAFLFDGRR LRAEQTPDELEMEDGDEIDAMLHQTGG (bdSENP1aminoacids248-481) SEQIDNO:11 PFVPLTDEDEDNVRHALGGRKRSETLSVHEASNIVITREILQCLNDKEWL NDEVINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRRW TTKRKLGYNLIDCDKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMK ALRILAKYLVDEVKDKSGKQIDVHAWKQEGVQNLPLQENGWDCGMFMLKY IDFYSRDMELVFGQKHMSYFRRRTAKEILDLKAG (bdNEDD8;BrachypodiumdistachyonNEDD8) SEQIDNO:12 MIKVKTLTGKEIEIDIEPTDTIDRIKERVEEKEGIPPVQQRLIYAGKQLA DDKTAKDYNIEGGSVLHLVLALRGG (bdNEDP1;BrachypodiumdistachyonNEDP1) SEQIDNO:13 MDERVLSYGDVVLLRSDLAILRGPHFLNDRIIAFYLAHLSASFHGDGDLL LLPPSIPYLLSNLPDPESVAEPLCLASRRLVLLPVNDNPDASVANGGSHW TLLVLDAATTDPQAPRFVHHDSLRGSANAAAARRLARALTAGGAPLRFVE APTPTQRNGHDCGVYVLAVARAICGWWRSSRRRENQQGGGGDWFATMMEE VDAESVGAMRAELLQLIHRLIQDKEQEEEKKSKAGVEDTCGQ (TEVproteaserecognitionsite-spacerfusion(the spacerbeingunderlined);cf.FIG.10A) SEQIDNO:14 ENLYFQGT (TEVprotease;TobaccoetchvirusNlaprotease) SEQIDNO:15 GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRR NNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKF REPQREERICLVTINFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQ CGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVS GWRLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ (TEV(SH)C6) SEQIDNO:16 ESLFKGPRDYNPISSSICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRN NGTLLVQSLHGVFKVKDTTTLQQHLVDGRDMIIIRMPKDFPPFPQKLKFR EPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQC GSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSG WRLNADSVLWGGHKVFMNKPEEPFQPVKEATQLMN (xlUb;Xenopuslaevisubiquitin) SEQIDNO:17 MQIFVKILTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQL EDGRTLSDYNIQKESTLHLVLRLRGG (xIUsp2;Xenopuslaevisubiquitin-specificpro- cessingprotease2) SEQIDNO:18 MRSHTLRIHGMGAGREHQIPGTVILSSIMDFILHRAKSSKHVQGLVGLRN LGNTCFMNSILQCLSNTKDLRDYCQQNSYRRDLSSKKCNTAIMEEFARLL QAIWTSSANEVVSPSEFKTQIQRYAPRFMGYNQQDAQEFLRFLLDGLHNE VNRVTVKPRPSSQDLDHMPDSEKGKKMWKRYLEREDSRIVELFVGQLKSS LTCTDCGYCSTVFDPFWDLSLPIAKKSASEVSLVDCMRLFTKEDVLDGDE KPTCCRCKARRRCTKKFTIQRFPKILVLHLKRFSEGRIRSGKLSTFVNFP LKDLDLREFSSESNPHATYNLYAVSNHSGTTMGGHYTAYCKNPSNGEWYT FNDSRVTAMSSSQVKSSDAYVLFYELSGPSSRM (HomosapiensAtg4B(hsAtg4B)) SEQIDNO:19 MDAATLTYDTLRFAEFEDFPETSEPVWILGRKYSIFTEKDEILSDVASRL WFTYRKNFPAIGGTGPTSDTGWGCMLRCGQMIFAQALVCRHLGRDWRWTQ RKRQPDSYFSVLNAFIDRKDSYYSIHQIAQMGVGEGKSIGQWYGPNTVAQ VLKKLAVFDTWSSLAVHIAMDNTVVMEEIRRLCRTSVPCAGATAFPADSD RHCNGFPAGAEVTNRPSPWRPLVLLIPLRLGLTDINEAYVETLKHCFMMP QSLGVIGGKPNSAHYFIGYVGEELIYLDPHTTQPAVEPTDGCFIPDESFH CQHPPCRMSIAELDPSIAVGFFCKTEDDFNDWCQQVKKLSLLGGALPMFE LVELQPSHLACPDVLNLSLDSSDVERLERFFDSEDEDFEILSL (HomosapiensLC3B(hsLC3B)) SEQIDNO:20 MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKT KFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYE SERDEDGFLYMVYASQETFG (HomosapiensGATE16(hsGATE16)) SEQIDNO:21 MKWMFKEDHSLEHRCVESAKIRAKYPDRVPVIVEKVSGSQIVDIDKRKYL VPSDITVAQFMWIIRKRIQLPSEKAIFLFVDKTVPQSSLTMGQLYEKEKD EDGFLYVAYSGENTFG (His.sub.14-SUMOstar-MBP) SEQIDNO:22 MSKHHHHSGHHHTGHHHHSGSHHHTGSDSEVNQEAKPEVKPEVKPETHIN LKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLTFLYDGIEIQAD QTPEDLDMEDNDIIEAHREQIGGAGTKTEEGKLVIWINGDKGYNGLAEVG KKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG LLAEITPDKAFQDKLYPFTINDAVRYNGKLIAYPIAVEALSLIYNKDLLP NPPKTWEEIPALDKELKAKGKSALMENLQEPYFTWPLIAADGGYAFKYEN GKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETA MTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNK ELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMEN AQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNGTGC (SUMOstar) SEQIDNO:23 MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLME AFAKRQGKEMDSLTFLYDGIEIQADQTPEDLDMEDNDIIEAHREQIGG (SUMOstarprotease) SEQIDNO:24 LVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPRRWLNDT IIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKTQIDKLD KIFTPINLNQSHWALGIIDLKKKTIGYVDSLSNGPNAMSFAILTDLQKYV MEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFDYK DAIRMRRFIAHLILTDALK (His.sub.14-IF2d1-xlLC3B-MBP) SEQIDNO:25 MSKHHHHSGHHHTGHHHHSGSHHHTGGSSGTDVTIKTLAAERQTSVERLV QQFADAGIRKSADDSVSAQEKQTLIDHLNQKNSGPDKLTLQRKTRSTLNI PGTGGKSKSVQIEVRKKRTFVKRDPQEAERLAAEEQAQREAEEQARREAE ESAKREAQQKAEREAAEQAKREAAEQAKREAAEKDKVTSSEKTFKQRRSL EQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSEL IKIIRRRLQLNSNQAFFLLVNGHSMVSVSTPISEVYEREKDEDGFLYMVY ASQETFGAGTKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVIVEHP DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLY PFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELK AKGKSALMFNLQEPYFTINPLIAADGGYAFKYENGKYDIKDVGVDNAGAK AGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQTNGTGC
EXAMPLES
[0208] Methods
[0209] Protein sequence alignments were performed using the ClustalW algorithm implemented in Protean version 11.2.1. (DNAStar, Inc.).
[0210] Substrate proteins and proteases were over-expressed in E. coli strain NEB Express from appropriate low copy expression vectors harboring an ColE1 origin of replication and conferring Kanamycin resistance as described before (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105). Further sequences are provided on request. Briefly, to produce protease substrates containing MBP as a target protein, E. coli cultures containing the appropriate expression vectors were grown with vigorous shaking over night at 30 C. in 50 ml TB medium containing 50 g/ml Kanamycin (TB-Kan). Cultures were diluted by addition of 300 ml fresh TB-Kan medium and further shaken at 30 C. After 30 min, expression of substrate proteins was induced by addition of IPTG to a final concentration of 200 M. After 3-4 h, 5 mM EDTA and 1 mM PMSF were added directly to the culture and cells were harvested by centrifugation for 7 min at 5000 g. The cell pellet was resuspended in ice-cold LS buffer (280 mM NaCl, 45 mM Tris/HCl pH 7.5, 4.5 mM MgCl.sub.2, 10 mM DTT) containing 15 mM imidazole at a final density of 100 OD.sub.600. After cell-lysis by sonication, cell debris were removed by centrifugation for 1 h at 200 000g. The supernatant was incubated with 2 ml of an EDTA- and DTT-resistant Ni.sup.2+-chelate resin (e.g. Roche cOmplete His-Tag Purification Resin) pre-equilibrated with LS buffer containing 15 mM imidazole for 1 h at 4 C. After washing off unbound proteins with LS buffer containing 15 mM imidazole, polyHis-tagged substrate proteins were eluted with LS buffer containing 300 mM imidazole. After exchanging the buffer to LS buffer using a PD-10 column (GE Healthcare), the substrate protein was mixed with 1/9 volume 2.5 M sucrose. Aliquots were snap-frozen in liquid nitrogen and stored at 80 C. until used.
[0211] Protease substrates with fluorescent target proteins (GFP or mCherry), and proteases were produced analogously after expression for 14-16 h at 18 C.
[0212] To obtain tag-free protease preparations, imidazole eluates were cleaved to completion with a polyHis-tagged protease appropriate for removal of the polyHis tag. After gel filtration on a SD200 16/60 column (GE Healthcare) pre-equilibrated with LS buffer, remaining traces of cleaved polyHis-tag and polyHis-tagged protease were removed by reverse Ni.sup.2+ chelate chromatography. This guaranteed the final enzyme preparation to be free of any contaminating proteolytic activity. Final protease preparations were diluted with 1/9 volume 2.5 M sucrose. Aliquots ere snap-frozen in liquid nitrogen and stored at 80 C. until used. All proteins were quantified via their absorption at 280 nm and computed extinction coefficients. Accuracy of quantification and purity of the proteins were validated by SDS-PAGE followed by Coomassie-staining.
[0213] Cleavage assays in solution and on column were performed as described before (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105; Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 106-115): If not stated otherwise, cleavage reactions were performed in LS-S buffer (250 mM NaCl, 40 mM Tris/HCl pH 7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 2 mM DTT). Generally, substrates and proteases were pre-diluted in LS-S buffer to twice the aspired end-concentration. Cleavage was initiated by mixing identical volumes (generally 5 l) of substrate and protease pre-dilutions and stopped by mixing with 9 volumes of hot SDS sample buffer. A fraction corresponding to 2.5 pg of substrate was separated by SDS-PAGE on 7-15% gradient gels. Gels were stained with Coomassie G250 and scanned.
[0214] On-column cleavage assays were done on EDTA- and DTT-resistant silica- or Sepharose-based Ni.sup.2+ chelate resins with high porosity.
[0215] Dynamic light scattering (DLS): Proteases diluted to 10 M in LS-S buffer were ultracentrifuged (200 000 g, 30 min), and assayed in a closed cuvette using a DynaPro NanoStar DLS instrument (Wyatt Technology). To acquire heat denaturation curves, the temperature was automatically raised by 1 C. every 10 min. DLS signals were acquired just before each temperature step.
[0216] In vitro binding assays: An EDTA- and DTT-resistant Ni.sup.2+ chelate resin was loaded with 40 M His.sub.14-Spacer-xlLC3B-GFP or His.sub.14-Spacer-xlGATE16-GFP. An empty resin served as a control. 20 l aliquots were incubated with 100 l of an equimolar mixture of full-length protease and a protease fragment (10 M each) for 1 h at 25 C. in LS-S buffer. After washing (330 sec) with the same buffer, bound proteins were eluted with SDS sample buffer containing 500 mM imidazole and analyzed by SDS-PAGE.
[0217] Example purifications from E. coli: Relevant fusion proteins were over-expressed from appropriate expression vectors in E. coli (ColE1 origin, Kanamycin resistance). Cleared lysates in LS buffer containing 15 mM imidazole were incubated with an EDTA- and DTT-resistant Ni.sup.2+ chelate resin. After washing with the same buffer, the target proteins were eluted with 500 nM xlAtg4B.sup.14-384 in LS buffer at 4 C. After 1 h, proteins remaining on the resin were eluted with LS buffer containing 0.5 M imidazole. Relevant fractions were analyzed by SDS-PAGE.
[0218] Samples taken during elution were in addition quantified by measuring the OD.sub.280. Substrate stability in eukaryotic extracts:
[0219] Rabbit reticulocyte lysate was purchased from Promega, wheat germ extract was prepared according to (Cathrin Enke, Doktorarbeit 2010, Cuvillier Verlag Gottingen, ISBN 978-3-86955-483-9), low-speed Xenopus egg extract was prepared according to (Blow, J. J., Laskey, R. A. (1986) Cell 47, 577-587). 1.25 l of 10 M protease substrates containing MBP as a target protein in LS-S buffer were incubated with 10 l of indicated lysates in the presence or absence of a protease mix containing scUlp1, SUMOstar protease, xlAtg4B.sup.14-384 and trAtg4B (0.1 M each final concentration, supplied in 1.25 l LS-S buffer) for 2 h at 25 C. in 12.5 l total volume. Reaction products were analyzed by Western blot with an antibody recognizing E. coli MBP (Sigma-Aldrich # M1321).
[0220] Yeast expression: S. cerevisiae strain SFY122 (S288C, Mata, H2B-CFP::TRP1, his3200, leu20, lys20, met150, ura30) was transformed with 2 expression plasmids encoding N-terminally ZZ-UBL-tagged Citrine (Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. and Tsien, R. Y. (2001) J Biol Chem 276, 29188-29194; Heikal, A. A., Hess, S. T., Baird, G. S., Tsien, R. Y. and Webb, W. W. (2000) Proc Natl Acad Sci U S A 97, 11996-12001) under the control of the GAL1 promoter (Sequences are provided on request). Single colonies were grown over night in CSM-Ura containing 2% glucose and 2% raffinose. Cells were washed three times in CSM-Ura +2% raffinose, diluted to OD.sub.600=0.2 and shaken over night at 30 C. Protein expression was induced by addition of 2% galactose for 5 h. Total lysates were prepared by the NaOH/TCA method (modified from (Riezman, H., Hase, T., van Loon, A. P., Grivell, L. A., Suda, K. and Schatz, G. (1983) EMBO J 2, 2161-2168)) and analyzed by Western blot using an antibody recognizing Citrine and CFP. The ZZ-tag was detected using a fluorescently labeled anti-mouse-IgG antibody.
[0221] For protein purifications from yeast, cells extracts were prepared by glass bead lysis (modified from (Conzelmann, A., Riezman, H., Desponds, C. and Bron, C. (1988) EMBO J 7, 2233-2240)) in LS-S buffer with protease protection. After centrifugation for 1 h at 200 000 g, cleared lysates were incubated with an anti-ZZ affinity resin. Non-bound material was washed off with LS-S buffer and target proteins were eluted with the appropriate protease in the same buffer within 1 h at 4 C. Material remaining on the resin was analyzed after elution with SDS sample buffer.
Example 1
Expression of xlLC3B-Fusions in E. coli
[0222] Initially, the primary aim was to analyze the suitability of xlAtg4B for tag removal from recombinant proteins fused to Xenopus laevis Atg8 orthologs. As the inventors had observed before that fusions to S. cerevisiae Atg8 only show suboptimal expression levels (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105), the inventors first compared the impact of various UBLs including xlLC3B on expression and solubility of GFP (
Example 2
Identification and Characterization of xlAtg4B Protease and xlAtg4B Protease Fragments
[0223] As a next step, the inventors wanted to find well-expressible and well-soluble xlAtg4B fragments displaying optimal stability and catalytic properties. Based on known structures of the human Atg4B homolog (Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Biol 355, 612-618; Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065; Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350), full-length xlAtg4B (residues 1-384) and five shorter xlAtg4B fragments harboring N- and/or C-terminal truncations (xlAtg4B.sup.14-384, xlAtg4B.sup.25-384 xlAtg4B.sup.1-345, xlAtg4B.sup.14-345 and xlAtg4B.sup.25-345) were cloned and expressed. All proteases fragments could be over-expressed in E. coli and obtained in high yield and purity (
[0224] To compare their catalytic properties efficiencies, a subset of these protease fragments was assayed in various in vitro cleavage assay (Frey, S. and GOrlich, D. (2014) J Chromatogr A 1337, 95-105) using two analogous substrate proteins with different xlAtg4B protease recognition sites (xlLC3B or xlGATE16, respectively;
[0225] Salt Sensitivity
[0226] To learn more about the nature of the possible interaction between xlAtg4B and its substrates xlLC3B and xlGATE16, the inventors next tested the salt sensitivity of substrate processing. To this end, each substrate was incubated with 500 nM of each protease for 1 h at 0 C. at defined salt concentrations (
[0227] Temperature Dependence
[0228] Next, the temperature dependence of substrate processing by the xlAtg4B fragments was analyzed (
[0229] xlAtg4B C-terminus
[0230] The results herein thus far show that deletion of the C-terminal xlAtg4B extension significantly impairs substrate processing, especially when using the xlGATE16 substrate at higher temperatures or elevated NaCl concentrations. Three possible explanations could possibly account for these effects: (i) The C-terminus of xlAtg4B might be required for enzymatic turnover, (ii) it could contribute to substrate recognition or (iii) be required for xlAtg4B stability. In the following, these scenarios were tested individually.
[0231] First, the cleavage efficiency of the xlAtg4B fragments at different dilutions was analyzed (
[0232] At high concentrations, also the xlGATE16 substrate was efficiently cleaved by the full-length protease or both N-terminally truncated fragments (
[0233] To directly compare binding of N- and/or C-terminally shortened protease fragments with the full-length enzyme, competitive pull-down assays using equimolar binary protease mixtures as a prey were performed (
[0234] Together, the experiments so far clearly show that xlAtg4B's C-terminal extension substantially contributes to recognition of both xlLC3B and xlGATE16 and is therefore required for robust substrate cleavage.
[0235] Thermal Stability
[0236] The inventors next asked if the N- and C-terminal extensions influence the (thermal) stability of xlAtg4B. To this end, all xlAtg4B fragments were pre-incubated for 16 h at different temperatures before analyzing their remaining activity in a standard xlLC3B cleavage assay (
[0237] In a second assay, dynamic light scattering (DLS) was used to analyze the thermal denaturation of the xlAtg4B fragments (
[0238] Interestingly, the temperatures required to observe an initial decline of enzymatic activity (
[0239] Promiscuity for Residues in the P.sub.1 Position
[0240] The experiments herein show that xlAtg4B.sup.14-384 combines optimal enzyme stability with efficient and robust substrate cleavage. The inventors further analyzed the properties of this protease fragment with respect to in-vitro cleavage of recombinant proteins. If target proteins with a defined (e.g. the authentic) N-terminus are to be produced, the enzyme's sensitivity to the residue in the P.sub.1 position (i.e. the residue following the scissile bond;
[0241] Discussion
[0242] Based on the known structure of the human Atg4B ortholog (hsAtg4B) (Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Blot 355, 612-618; Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065; Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350), a series xlAtg4B fragments with N-terminal and C-terminal truncations was designed. At low temperature, the analyzed N-terminally truncated xlAtg4B fragments (xlAtg4B.sup.14-384 and xlAtg4B.sup.25-384) showed a catalytic activity comparable to the full-length enzyme. While these results seem to be in contrast to earlier studies on the human Atg4B ortholog that suggested an auto-inhibitory function of the N-terminal extension (Li, M., Hou, Y., Wang, J., Chen, X., Shao, Z. M. and Yin, X. M. (2011) J Biol Chem 286, 7327-7338; Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350), the inventors observed that at temperatures 16 C., indeed the two shorter fragments were slightly more active than the full-length enzyme. Without being bound by theory, this temperature effect could potentially be a result of several hydrophobic interactions that are observed between the N-terminal extension of the human enzyme and the protease surface near the catalytic center. Importantly, deletion of only 13 N-terminal residues was sufficient to is efficiently prevent auto-inhibition and in addition created an enzyme fragment (xlAtg4B.sup.14-384) with superior temperature stability compared to xlAtg4B.sup.25-384.
[0243] The contribution of the flexible C-terminal protease extension (residues 346-384) to substrate recognition and processing was also analyzed. While the significance of this region so far had not been directly addressed, it was now found compelling evidence that it is required for an efficient interaction with two dedicated xlAtg4B substrate proteins, xlLC3B and xlGATE16. This finding was surprising for two reasons. First, the available structures of the substrate-free human Atg4B (hsAtg4B) suggest that the protease's C-terminus partially occupies the substrate-binding site (Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Biol 355, 612-618; Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065). It therefore has to be displaced before substrate binding can occur, which may thus hamper formation of the proteasesubstrate complex. Second, crystals of LC3B-bound hsAtg4B could be obtained only after removal of the C-terminal extension (Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350), which (i) shows that the C-terminus of xlAtg4B is not strictly required for substrate interaction and (ii) could indeed suggest an inhibitory effect on complex formation. In contrast, the results herein clearly show that the C-terminal extension is an integral part of the protease's substrate interaction surface.
[0244] While the C-terminal protease truncation affects processing of xlLC3B mainly under stringent conditions (high salt, elevated temperature or low substrate concentration), the effect is pronounced already under standard conditions (0 C., 250 mM NaCl, 100 M initial substrate concentration) when using the xlGATE16 substrate. This striking difference might be attributed to the overall lower affinity of xlGATE16 to xlAtg4B as compared to xlLC3B (see
[0245] In combination, the folded core and the C-terminal extension of xlAtg4B mediate a strong interaction with the xlLC3B substrate, which is beneficial for efficient substrate processing at high dilution and complete processing of substrates. Strikingly, however, the turnover rate at high substrate concentrations is clearly lower for xlLC3B as compared to the xlGATE16 substrate, which has a lower affinity to the protease. Without being bound by theory, this seemingly paradoxical finding suggests that the rate-limiting step in xlAtg4B-mediated substrate cleavage is the substrate release. As a consequence, cleavage of xlLC3B substrates is slower but more robust.
Example 3
Application of the xlAtg4B Protease System for Tag Removal and On-Column Cleavage
[0246] An important application of tag-cleaving proteases is on-column cleavage of recombinant proteins. The inventors directly addressed the suitability of xlAtg4B.sup.14-384 for this purpose using polyHis-tagged substrate proteins bound to a Silica-based Ni.sup.2+ chelate resin of high porosity (
[0247] An important parameter for the practical application of tag-cleaving proteases is their substrate specificity. This parameter is especially important when mutually exclusive specificity (orthogonality) to other proteases is strictly required, e.g. for purification of protein complexes with controlled subunit stoichiometry (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 106-115). Also, it is important to know which host proteases could potentially cleave a given protease recognition site during expression. For practical applications, the inventors were especially interested in the cross-reactivity of xlAtg4B with the well-established TEV protease (Kapust, R. B., et al. (2001) Protein Engineering 14(12), 993-1000; van den Berg, S., et al. (2006) Journal of Biotechnology 121, 291-298), scUlp1 (Malakhov, M. P., et al. (2004) J Struct Funct Genomics 5, 75-86), SUMOstar protease (Liu, L., et al. (2008) Protein Expr Purif 62, 21-28; Peroutka, R. J., et al. (2008) Protein Sci 17, 1586-1595) and the recently described proteases bdSENP1, bdNEDP1, and xlUsp2 (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105; Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 106-115). In addition, the wheat (Triticum) Atg4 ortholog (trAtg4) was also included. To analyze the specificity profiles of these proteases, a high concentration (20 M) of each protease was incubated with 100 M of each substrate protein (see
[0248] These inter- and intra-species substrate preferences of Atg4-like enzymes were analyzed further using detailed protease titration assays (
[0249] The best performing xlAtg4B fragment, xlAtg4B.sup.14-384, has great potential as a new tag-cleaving protease. This protease fragment is highly active and routinely cleaves a 100- to 200-fold substrate excess within 1 h at 0 C. For comparison, TEV protease, which is probably still the most common tag-cleaving protease, requires 30- to 50-fold higher protease concentrations under these conditions (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105). In addition, xlAtg4B.sup.14-384 is highly temperature stable (42 C. for 16 h) and can therefore also be used at higher temperatures. At 25 C., e.g., xlAtg4B.sup.14-384 can cleave a 2 000-fold substrate excess within one hour; at 37 C. even less protease is required for efficient cleavage. When used for in-vitro tag removal from recombinant proteins, this high specific activity reduces contamination of the final protein preparation by the protease. Compared to other tag-cleaving proteases like yeast Ulp1p (Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D. and Butt, T. R. (2004) J Struct Funct Genomics 5, 75-86; Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105) or SUMOstar protease (Liu, L., Spurrier, J., Butt, T. R. and Strickler, J. E. (2008) Protein Expr Purif 62, 21-28), xlAtg4B displays a superior salt tolerance (tested up to 1.5 M NaCl) and a broad P.sub.1 promiscuity, parameters that are important for robust cleavage of recombinant substrate proteins in various buffer conditions and sequence contexts.
[0250] When analyzing cross-reactivity with other tag-cleaving proteases, the inventors found out that xlAtg4B displays orthogonal specificity to the recently introduced bdSENP1 and bdNEDP1 proteases (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105). Together, these highly efficient proteases thus ideally complement each other and can be combined to purify protein complexes with is controlled subunit stoichiometry by successive affinity capture and proteolytic release steps (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 106-115).
Example 4
Application of the xlAtg4B Protease System for Purification of Proteins from Eukaryotic Hosts
[0251] The unexpectedly high resistance of xlLC3B towards cleavage by Atg4-like proteases originating from other species encouraged to address the stability of xlLC3B- and xlGATE16 fusions in various eukaryotic cell extracts (
[0252] Next, it was desired to find out if some of the analyzed ubiquitin-like protease recognition sites would also be compatible with production of intact full-length recombinant fusion proteins in a living eukaryotic host. Therefore different ZZ-UBL-Citrine substrate proteins (
[0253] To show that the xlLC3B/xlAtg4B and bdNEDD8/bdNEDP1 systems are indeed suited for purification of recombinant proteins from a eukaryotic host, recombinant Citrine was purified as a model target protein from S. cerevisiae. To this end, the ZZ-UBL-Citrine fusions were over-expressed in yeast for 5 h as before. After cell lysis in a native buffer, the full-length fusion protein was found in the soluble fraction from which highly pure recombinant Citrine could be obtained by an efficient one-step capture and on-column cleavage procedure (
[0254] Discussion
[0255] Importantly, both analyzed xlAtg4B substrates, xlLC3B and xlGATE16, promote solubility and high-level expression of the respective fusion proteins in E. coli (see
[0256] In addition, xlLC3B features additional remarkable advantages: It was found that xlLC3B fusions are stable in wheat germ extract and even under drastic conditions only marginally processed by wheat Atg4 (trAtg4) in vitro, suggesting that stable xlLC3B fusion proteins can be produced in plants. Even more, xlLC3B is not recognized by the S. cerevisiae Atg4 protease. Full-length xlLC3B fusions can thus be expressed in this eukaryotic host and purified by a simple one-step capture and proteolytic release strategy. Such eukaryotic expression might be exploited for the production of proteins that rely on the eukaryotic folding machinery or have to be modified by posttranslational modifications. Fully unexpectedly, it was found that also bdNEDD8 fusion proteins are only marginally processed in yeast. With xlLC3B, bdNEDD8 (Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105) and SUMOstar (Liu, L., Spurrier, J., Butt, T. R. and Strickler, J. E. (2008) Protein Expr Purif 62, 21-28; Peroutka, R. J., Elshourbagy, N., Piech, T. and Butt, T. R. (2008) Protein Sci 17, 1586-1595), there are now three orthogonal UBL-derived protease recognition sites that in principle allow for full-length protein production in S. cerevisiae (
LIST OF REFERENCES
[0257] WO 2015/049230
[0258] WO 2008/083271 A2
[0259] WO 2002/090495 A2
[0260] WO 2003/057174 A2
[0261] WO 2005/003313 A2
[0262] WO 2006/073976 A2
[0263] Hemelaar, J., Lelyveld, V. S., Kessler, B. M. and Ploegh, H. L. (2003) J Biol Chem 278, 51841-51850
[0264] Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y. and Yoshimori, T. (2004) J Cell Sci 117, 2805-2812
[0265] Marino, G., Uria, J. A., Puente, X. S., Quesada, V., Bordallo, J. and Lopez-Otin, C. (2003) J Biol Chem 278, 3671-3678
[0266] Tanida, I., Sou, Y. S., Ezaki, J., Minematsu-Ikeguchi, N., Ueno, T. and Kominami, E. (2004) J Biol Chem 279, 36268-36276
[0267] Li, M., Hou, Y., Wang, J., Chen, X., Shao, Z. M. and Yin, X. M. (2011) J Biol Chem 286, 7327-7338
[0268] Woo, J., Park, E. and Dinesh-Kumar, S. P. (2014) Proc Natl Acad Sci U S A 111, 863-868
[0269] Kumanomidou, T., Mizushima, T., Komatsu, M., Suzuki, A., Tanida, I., Sou, Y. S., Ueno, T., Kominami, E., Tanaka, K. and Yamane, T. (2006) J Mol Biol 355, 612-618
[0270] Sugawara, K., Suzuki, N. N., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2005) J Biol Chem 280, 40058-40065
[0271] Satoo, K., Noda, N. N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y. and Inagaki, F. (2009) EMBO J 28, 1341-1350
[0272] van der Veen, A. G. and Ploegh, H. L. (2012) Annu Rev Biochem 81, 323-357
[0273] Yeh, E. T., Gong, L. and Kamitani, T. (2000) Gene 248, 1-14
[0274] Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D. and Butt, T. R. (2004) J Struct Funct Genomics 5, 75-86
[0275] Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 95-105
[0276] Frey, S. and Grlich, D. (2014) J Chromatogr A 1337, 106-115
[0277] Liu, L., Spurrier, J., Butt, T. R. and Strickler, J. E. (2008) Protein Expr Purif 62, 21-28
[0278] Peroutka, R. J., Elshourbagy, N., Piech, T. and Butt, T. R. (2008) Protein Sci 17, 1586-1595
[0279] Taxis, C. and Knop, M. (2012) Methods Mol Biol 832, 611-626
[0280] Urabe, M., Kume, A., Takahashi, T., Serizawa, N., Tobita, K. and Ozawa, K. (1999) Biochem Biophys Res Commun 266, 92-96
[0281] Taxis, C., Stier, G., Spadaccini, R. and Knop, M. (2009) Mol Syst Biol 5, 267
[0282] Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. and Tsien, R. Y. (2001) J Biol Chem 276, 29188-29194
[0283] Heikal, A. A., Hess, S. T., Baird, G. S., Tsien, R. Y. and Webb, W. W. (2000) Proc Natl Acad Sci U S A 97, 11996-12001
[0284] Riezman, H., Hase, T., van Loon, A. P., Grivell, L. A., Suda, K. and Schatz, G. (1983) EMBO J 2, 2161-2168
[0285] Conzelmann, A., Riezman, H., Desponds, C. and Bron, C. (1988) EMBO J 7, 2233-2240
[0286] Butt, T. R., Edavettal, S. C., Hall, J. P. and Mattern, M. R. (2005) Protein Expr Purif 43, 1-9
[0287] Arnau, J., Lauritzen, C., Petersen, G. E. and Pedersen, J. (2006) Protein Expr Purif 48, 1-13
[0288] Li, S. J. and Hochstrasser, M. (1999) Nature 398, 246-251
[0289] Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. and Nygren, P. A. (1997) Protein Expr Purif 11, 1-16
[0290] Young, C. L., Britton, Z. T. and Robinson, A. S. (2012) Biotechnol J 7, 620-634
[0291] Renicke, C., Spadaccini, R. and Taxis, C. (2013) PLoS One 8, e67915 UniProt Q640G7
[0292] Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E. (2001) Protein Engineering 14(12), 993-1000
[0293] van den Berg, S., Lofdahl, P.A., Hard, T., Berglund, H. (2006) Journal of Biotechnology 121, 291-298.
[0294] Cathrin Enke, Doktorarbeit 2010, Cuvillier Verlag Gottingen, ISBN 978-3-86955-483-9
[0295] Blow, J. J., Laskey, R. A. (1986) Cell 47, 577-587.