SELECTION SYSTEM FOR EVOLVING PROTEASES AND PROTEASE-CLEAVAGE SITES

Abstract

The present invention relates to a fusion protein, comprising the structure N-PCS.sup.Y-degSig.sub.N-M-PCS.sup.X-degSig.sub.CC; wherein N represents the N-terminus; PCS.sup.Y and PCS.sup.X each represent a protease cleavage site (PCS), which differ from each other in at least one amino acid residue; degSig.sub.N represents a degradation signal which promotes degradation of the fusion protein in a host pot cell if PCS.sup.Y is cleaved by a protease such that the first amino acid of degSig.sub.N becomes the new N-terminus of the remaining fusion; M represents a cytoplasmic selection marker; and degSig.sub.C represents a second degradation signal which promotes degradation of the fusion protein in a host cell if PCS.sup.X is not cleaved by a protease; and C represents the C-terminus. Further provided is a nucleic acid construct, comprising a nucleic acid sequence coding for said fusion protein, a nucleic acid expression construct library, comprising a plurality of such nucleic acid expression constructs in diversified form, and methods using the fusion protein and nucleic acid constructs coding therefor. Finally, the present invention provides variants of bdSUMO and bdSENP1 which have been identified by the methods of the present disclosure, and which exhibit improved properties over existing orthogonal protease/protease cleavage site-pairs which are currently used with wild-type bdSUMO and wildtype bdSENP1.

Claims

1-24. (canceled)

25. A variant protease, wherein said variant protease cleaves a protease cleavage site (PCS) having the amino acid sequence of SEQ ID NO: 41 (Mut1 bdSUMO) and fused to the N-terminus of MBP having the amino acid sequence of SEQ ID NO: 71 more efficiently after the C-terminal Gly-Gly motif than a protease cleavage site having the amino acid sequence of SEQ ID NO: 4 (scSUMO) fused to the N-terminus of SEQ ID NO: 71 or a protease cleavage site having the amino acid sequence of SEQ ID NO: 3 (hsSUMO2) fused to the N-terminus of SEQ ID NO: 71, when tested at the same concentration under standard conditions of 1 hour incubation at 21° C., an initial concentration of PCS-MBP fusions of 100 μM in a buffer consisting of 45 mM Tris/HCl pH 7.5, 250 mM NaCl, 2 mM MgCl.sub.2, 250 mM sucrose, 10 mM DTT.

26. The variant protease of claim 25, wherein said variant protease cleaves an at least a 500-fold molar excess of the Mut1 bdSUMO-MBP fusion at the above standard conditions.

27. The variant protease of claim 25, wherein said variant protease has at least 80% sequence identity over the full-length of SEQ ID NO: 6 (bdSENP1), wherein said variant protease, when aligned to the full-length sequence of SEQ ID NO: 6, comprises a substitution at the position corresponding to N280 of the aligned 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.

28. The variant protease of claim 27, wherein the amino acid is selected from the group consisting of S, H, Q, and A.

29. The variant protease of claim 27, wherein said variant protease, when aligned to the full-length sequence of SEQ ID NO: 6, further comprises a substitution at the position corresponding to R356 of the aligned SEQ ID NO: 6, wherein the amino acid at said position is substituted by another amino acid selected from the group consisting of E, S, V, Y, and L.

30. The variant protease of claim 29, wherein the substitution is selected from the group consisting of E, S, and V.

31. The variant protease of claim 29, wherein said variant protease, when aligned to the full-length sequence of SEQ ID NO: 6, further comprises a substitution at the position corresponding to R269 of the aligned SEQ ID NO: 6, wherein the amino acid at said position is substituted by another amino acid selected from the group consisting of E, S, P, K, and V.

32. The variant protease of claim 29, wherein said variant protease, when aligned to the full-length sequence of SEQ ID NO: 6, further comprises a substitution at the position corresponding to K350 of the aligned SEQ ID NO: 6, wherein the amino acid at said position is substituted by another amino acid selected from the group consisting of M, E, V, G, T, and R.

33. The variant protease of claim 32 wherein the substitution is selected from the group consisting of M, E, V, G and T.

34. The variant protease of claim 27, wherein said variant protease, when aligned to the full-length sequence of SEQ ID NO: 6, further comprises amino acid substitution(s) at two or three positions selected from the group of R356, R269 and K350 of the aligned SEQ ID NO:6, wherein the amino acid substitution at position R356 is a substitution by an amino acid selected from the group consisting of E, S, V, Y, and L, the amino acid substitution at position R269 is a substitution by an amino acid selected from the group consisting of E, S, P, K, and V, and the amino acid substitution at position K350 is a substitution by an amino acid selected from the group consisting of M, E, V, G, T, and R.

35. The variant protease of claim 27, wherein said variant protease comprises a combination of substitutions selected from the group consisting of (i) 280S, 346E (MutB); (ii) 280H, 269S, K350V (MutG); (iii) 269P, 280A, 346E, 350M (MutH); (iv) 269K, 280H, 346E, 350E (Muti); (v) 269E, 280S, 346S, 350T (MutJ); and (vi) 269V, 280Q, 346V, 350G (MutK).

36. The variant protease of claim 27 having the amino acid sequence of SEQ ID NO: 6 (bdSENP1), except for the substitution at position N280, optionally, if present, in combination with the additional substitutions at positions R269, R346, and/or K350.

37. The variant protease of claim 36 having the amino acid sequence of any one of SEQ ID NO: 56 to SEQ ID NO: 70.

38. A process of purifying a protein of interest, comprising the steps of (i) providing a protein of interest to be purified wherein said protein comprises an affinity tag fused to said protein via a variant SUMO protease cleavage site (PCS); (ii) binding the protein of step (i) to an affinity matrix via said affinity tag; and (iii) eluting the protein from the affinity matrix using a variant protease of claim 25; thereby purifying the protein; wherein said variant SUMO PCS comprises a C-terminal Gly-Gly SUMO motif, and, when fused to the N-terminus of MBP having the amino acid sequence of SEQ ID NO: 71, is cleaved more efficiently after the C-terminal Gly-Gly SUMO motif by a protease having the amino acid sequence of SEQ ID NO: 57 (MutB bdSENP1) as compared to cleavage by a protease having the amino acid sequence of SEQ ID NO: 7 (scUlp1) or SEQ ID NO: 8 (hsSENP2), when tested at the same concentration under standard conditions of 1 hour incubation at 21° C., an initial concentration of SUMO PCS-MBP fusion of 100 μM in a buffer consisting of 45 mM Tris/HCl pH 7.5, 250 mM NaCl, 2 mM MgCl.sub.2, 250 mM sucrose, 10 mM DTT.

39. The process of claim 38, wherein said variant SUMO PCS has at least 80% sequence identity over the full-length of SEQ ID NO: 3 (bdSUMO), or which is a homolog of the bdSUMO protease cleavage site shown in SEQ ID NO: 3, wherein said variant protease cleavage site, when aligned to the full-length sequence of SEQ ID NO: 3, comprises a substitution at the position corresponding to D67 of the aligned SEQ ID NO: 3, wherein the amino acid at said position is substituted by a another amino acid selected from the group consisting of K, R, N, A and H.

40. The process of claim 39, wherein said variant SUMO PCS, when aligned to the full-length sequence of SEQ ID NO: 3, further comprises: (a) a substitution at the position corresponding to Q75 of the aligned SEQ ID NO: 3, wherein the amino acid at said position is substituted by another amino acid selected from the group consisting of R, W, A, H, M, I, P, and F; or (b) a substitution at the position corresponding to T60 of the aligned SEQ ID NO: 3, wherein the amino acid at said position is substituted by a another amino acid selected from the group consisting of S, N, K, P, H, R, and Q; or (c) both of (a) and (b).

41. The process of claim 39, wherein, wherein said variant SUMO PCS comprises a combination of substitutions selected from the group consisting of (i) 67K, 60K, 75R (Mut1); (ii) 67K, 60P, 75W (Mut8); (iii) 67K, 75R (Mut10); (iv) 67K, 60S, 75H (Mut11); (v) 67K, 60S, 75W (Mut12); (vi) 67K, 60S, 75A (Mut13); (vii) 67K, 60N, 75W (Mut14); and (viii) 67K, 60N, 75A (Mut15).

42. The process of claim 39, wherein said variant SUMO PCS has the amino acid sequence of SEQ ID NO: 3 (bdSUMO), except for the substitution D67K.

43. The process of claim 42, wherein said variant SUMO PCS has additional substitutions at position T60, or at position Q75, or at both positions T60 and Q75.

44. The process of claim 42, wherein variant SUMO PCS has an amino acid sequence selected from the group consisting of SEQ ID NO: 41 to SEQ ID NO: 55.

Description

DESCRIPTION OF THE FIGURES

[0215] FIG. 1 Diagram of the in vivo system for the selection of proteases and protease-cleavage sites with orthogonal specificities. The system is based on the survival of E. coli cells in selective medium containing hygromycin B. Cells co-express a SUMO-specific protease and the fusion protein SUMO.sup.Y-Degron.sup.NER-HygB-SUMO.sup.X-ssrA as a selection marker. Cells survive only if a given SUMO-specific protease shows orthogonal specificity to two different protease cleavage sites (SUMO.sup.Y and SUMO.sup.X).

[0216] FIG. 2 Validation of the in vivo selection system. (A) The SUMO protein (bdSUMO) and the SUMO-specific protease 1 (bdSENP1) from B. distachyon were used as model proteins to the test the functionality of the selection system. A non-cleavable SUMO mutant (SUMO*) was used to account against cleavage. (B) Cells expressing a HygB construct lacking both protein degradation signals were used as positive control for cellular growth in selective media. (C) Only cells expressing SUMO*-Degron.sup.NER-HygB-bdSUMO-ssrA survived due to the permanent inactivation of both degradation signals. (D, E, C and F). Bacteria co-expressing bdSENP1 and a different reporter protein do not survive after the degradation of HygB due to activation of the Degron.sup.NER and/or the lack of inactivation of the ssrA signal.

[0217] FIG. 3 (A) Illustration of the fusion proteins used to select for bdSUMO mutants (bdSUMO.sup.MutX), which are not cleaved by the SUMOstar protease. (B) Sequence alignment of ten different bdSUMO mutants. Identical residues are highlighted in black boxes and the numbering of the sequence is according to the full-length wild type (wt) bdSUMO protein. The bdSUMO mutant that belongs to the SUMOvera system is underlined (bdSUMO.sup.Mut1).

[0218] FIG. 4 (A) Reporter construct used to select for bdSENP1 mutants (bdSENP1.sup.MutX) that cleave bdSUMO.sup.Mut1 but do not recognize SUMOstar as substrate. (B) Sequence alignment of the wild type bdSENP1 and six different functional bdSENP1 mutants. The bdSENP1 mutant that belongs to the SUMOvera system is shown underlined (bdSENP1.sup.MutB). The numbering of the sequencing is set according to the full-length bdSENP1 protein and identical residues are highlighted in black.

[0219] FIG. 5 Effect of each mutation in bdSUMO.sup.Mut1 for the cleavage resistance against SUMOstar protease. Different variants of the bdSUMO-MBP fusion protein containing one, two or three mutations as in bdSUMO.sup.Mut1 were incubated with a large amount of SUMOstar protease (10 μM) for 2h at 25° C. Samples were resolved by SDS-PAGE to separate the fusion protein from the C-terminal cleaved MBP. The same bdSUMO-MBP fusion proteins were used to evaluate the contribution of each mutation in bdSUMO.sup.Mut1 for the recognition by bdSENP1.sup.MutB. Samples containing 100 μM of each MBP fusion protein were incubated with 200 nM of bdSENP1.sup.MutB protease for 1h at 4° C.

[0220] FIG. 6 Proteolytic efficiency of the bdSENP1.sup.MutB protease and other site-specific proteases. The proteolytic efficiency of different site-specific proteases was tested in solution for 1h at 0° C. Different amount of a given protease were incubated with 100 μM of the corresponding tagged MBP. Samples were resolved by SDS-PAGE to separate the full-length fusion protein from the C-terminal cleaved MBP. Samples highlighted with a black box indicate the lowest protease concentration at which ≈95% of the cognate substrate is cleaved. The experiments are ordered in the figure from the most to the least efficient protease used in this experiment.

[0221] FIG. 7 bdSENP1.sup.MutB and SUMOstar proteases have fully orthogonal specificities. Samples containing 100 μM of a given H14-SUMO-MBP fusion protein were incubated with increasing concentration of either bdSENP1.sup.MutB or SUMOstar proteases for 1h at 0° C. The full-length SUMO-fusion protein was separated from the C-terminal cleaved MBP by SDS-PAGE. The asterisks represent the protease concentration at which ≈95% of the cognate SUMO-fusion protein is cleaved.

[0222] FIG. 8 bdSUMO.sup.Mut1-fusion proteins are stable in S. cerevisiae. (A) Scheme of the SUMO fusion proteins over-expressed in S. cerevisiae (H14: poly-histidine tag, ZZ: double copy of the Z-domain from staphylococcal protein A). (B) After 6h of protein over-expression at 30° C., the stability of different SUMO fusion proteins was analyzed by western blot using an anti-citrine antibody. Cyan fluorescent protein fused to histone 2B (H2B-CFP) was used as an internal control to confirm even loading of the samples. Cross-reactivity between the anti-citrine antibody and the ZZ-tag is indicated by *. An empty yeast lysate was used as a negative control.

[0223] FIG. 9 bdSUMO.sup.Mut1 is a stable fusion tag in eukaryotic lysates. (A) Figure of fusion proteins used to test the stability of different SUMO proteins in several eukaryotic lysates. (H14: poly-histidine tag, MBP: E. coli Maltose Binding Protein). (B) The stability of different SUMO fusion proteins was analyzed by western blot using an anti-MBP antibody after incubation in highly concentrated eukaryotic extracts for 2h at 30° C. The presence of a C-terminal cleaved MBP indicates the cleavage of the SUMO tag by the endogenous SUMO specific proteases. Samples with a protease mix (Ulp1, SUMOstar protease, bdSENP1 and bdSENP1.sup.MutB protease, 1 μM each) were included to rule out the presence of protease inhibitory substances present in the lysates.

[0224] FIG. 10 Purification of a stoichiometric protein complex in yeast. (A) As a model for a hetero-dimeric complex, an anti-citrine nanobody (Nb) tagged with bdSUMO.sup.Mut1 and SUMOstar-tagged citrine were used. (B) Scheme of the purification processes composed of two consecutive chromatographic steps. The dimeric complex was co-expressed in S. cerevisiae as a soluble form for 6h at 30° C. (soluble material). First, the complex was isolated through a poly-histidine tag (H14) fused to bdSUMO.sup.Mut1 using a Ni.sup.2+ chelate matrix (first chromatographic step). The complex was then eluated by in-column cleavage using bdSENP1.sup.MutB (On-column cleavage eluate 1). For the second purification step, the eluate 1 was loaded onto an anti-Z domain matrix to bind to the ZZ-tag fused to SUMOstar. The stoichiometric and highly pure complex was then finally eluted by on-column cleavage using the SUMOstar protease (On-column cleavage eluate 2). (C) Protein samples corresponding to 35 mOD units of cells or 1/1000 of the total purified protein were analyzed by SDS-PAGE and further stained by coomassie blue. Labels on the middle of both images side define the protein identity of each band in both gels.

[0225] FIG. 11 bdSENP1.sup.MutB protease can be ectopically over-expressed in S. cerevisiae. The viability of the yeast cells over-expressing a SUMO-specific protease was tested. Cells were subjected to different 10-fold dilutions (from 1:25 to 1:3125 v/v) and further spotted on plates containing glucose or galactose to repress and induce protein expression, respectively. Cells transformed with an empty vector or encoding a catalytically dead protease (C440S) were used as negative controls.

[0226] FIG. 12 (A) Analysis of the amino acid frequency in the three mutagenized resides (T60X, D67X and Q75X) for the all the bdSUMO mutants selected by phage display. (B) Sequence alignment of five different bdSUMO mutants that contain the most frequent mutations after selection by phage display. Identical residues are highlighted in black boxes and the numbering of the sequence is according to the full-length wild type (wt) bdSUMO protein.

[0227] FIG. 13 (A) Selection construct used in E. coli for the selection of bdSENP1 mutants (bdSENP1.sup.MutX) that cleavage preferentially bdSUMO.sup.Mut1 and not wild type scSUMO. (B) Sequence alignment of bdSENP1.sup.MutB and the six most abundant bdSENP1 mutants after selection using the construct described in A. The numbering of the residues is according to the full-length protein and the “-” denotes an amino acid deletion in the corresponding bdSENP1 mutant. Residues highlighted in black are strictly conserved within all mutants and the wild type (wt) bdSENP1 protease.

[0228] FIG. 14 Analogues mutations of the bdSUMO.sup.Mut1 system can be used in wild type SUMO/SUMO proteases systems. scSUMO-MBP and hsSUMO2-MBP fusion proteins, containing mutations at the equivalent positions to the ones in the bdSUMO.sup.Mut1 and bdSUMO.sup.Mut11, were incubated for 1h at 25° C. together with different SUMO-specific proteases. Numbering of the residues in scSUMO and hsSUMO2 is according to the full-length protein sequence. Samples were resolved by SDS-PAGE to separate the full-length fusion protein from the C-terminal cleaved MBP.

[0229] FIG. 15 Substrate specificity of different wild type and mutant SUMO-specific proteases. (A) Samples with 100 μM of MBP fused to a SUMO variant (wild type SUMO or different bdSUMO mutants) were incubated for 1h at 25° C. with different SUMO-specific proteases. The protease concentration used in each assay is sufficient to completely cleave the cognate SUMO protein at the conditions mentioned above. Samples corresponding to around 2 μg of the SUMO fusion were analyzed by SDS-PAGE to separate the full-length fusion protein from the cleaved MBP. (B, C) List of the mutations in the bdSUMO and SENP1 variants tested in A, respectively

[0230]

TABLE-US-00002 SEQUENCES SEQ ID NO: 1 (Degron.sup.NER) FLFVQ SEQ ID NO: 2 (ssrA) AADENYALAA SEQ ID NO: 3 (WT bdSUMO amino acids 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMT AIAFLFDGRRLRAEQTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 4 (WT scSUMO amino acids 1-98) MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEM DSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG SEQ ID NO: 5 (WT hsSUMO2; Homo sapiens SUMO2, amino acids 1-93) MADEKPKEGVKTENNDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQIRF RFDGQPINETDTPAQLEMEDEDTIDVFQQQTGG SEQ ID NO: 6 (WT bdSENP1 amino acids 248-491) PFVPLTDEDEDNVRHALGGRKRSETLSVHEASNIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRRWTTKRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVONLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 7 (scUlp1 amino acids 403-621) LVPELNEKDDDQVQKALA SRENTQLMNRDNIEITVRDFKTLAPRRWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLS ERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQSHWALGIIDLKKKTIGYVDSLSNGPNAM SFAILTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFD YKDAIRMRRFIAHLILTDALK SEQ ID NO: 8 (hsSENP2; Homo sapiens SENP2, amino acids 419-644) EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLNDEIINFYMNMLME RSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVD FRKKNITYYDSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMNG SDCGMFACKYADCITKDRPINFTQQHMPYFRKRMVWEILHRKLL SEQ ID NO: 9 (Wt bdSUMO; amino acids 56-79) SVDMTAIAFLFDGRRLRAEQTPDE SEQ ID NO: 10 (Mut1 bdSUMO; amino acids 56-79) SVDMKAIAFLFKGRRLRAERTPDE SEQ ID NO: 11 (Mut2 bdSUMO; amino acids 56-79) SVDMTAIAFLFKGRRLRAECTPDE SEQ ID NO: 12 (Mut3 bdSUMO; amino acids 56-79) SVDMHAIAFLFKGRRLRAEKT PDE SEQ ID NO: 13 (Mut4 bdSUMO; amino acids 56-79) SVDMRAIAFLFRGRRLRAEVTPDE SEQ ID NO: 14 (Mut5 bdSUMO; amino acids 56-79) SVDMTAIAFLFKGRRLRAEFTPDE SEQ ID NO: 15 (Mut6 bdSUMO; amino acids 56-79) SVDMHAIAFLFKGRRLRAEQTPDE SEQ ID NO: 16 (Mut7 bdSUMO; amino acids 56-79) SVDMDAIAFLFRGRRLRAECTPDE SEQ ID NO: 17 (Mut8 bdSUMO; amino acids 56-79) SVDMPAIAFLFKGRRLRAEWTPDE SEQ ID NO: 18 (Mut9 bdSUMO; amino acids 56-79) SVDMAAIAFLFKGRRLRAEYTPDE SEQ ID NO: 19 (Mut10 bdSUMO; amino acids 56-79) SVDMTAIAFLFKGRRLRAERTPDE SEQ ID NO: 20 (Mut11 bdSUMO; amino acids 56-79) SVDMSAIAFLFKGRRLRAEWTPDE SEQ ID NO: 21 (Mut12 bdSUMO; amino acids 56-79) SVDMSAIAFLFKGRRLRAEHTPDE SEQ ID NO: 22 (Mut13 bdSUMO; amino acids 56-79) SVDMSAIAFLFKGRRLRAEATPDE SEQ ID NO: 23 (Mut14 bdSUMO; amino acids 56-79) SVDMNAIAFLFKGRRLRAEWTPDE SEQ ID NO: 24 (Mut15 bdSUMO; amino acids 56-79) SVDMNAIAFLFKGRRLRAEATPDE SEQ ID NO: 25 (Wt bdSENP1 amino acids 265-354) GGRKRSETLSVHEASNIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK- CHFENTFFYKKLINGGYDYKSVRRWTTKRKLG SEQ ID NO: 26 (MutA bdSENP1 amino acids 265-354) GGRKPSETLSVHEASGIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK- CHFFNTFFYKKLINGGYDYKSVREWTTPRKLG SEQ ID NO: 27 (MutB bdSENP1 amino acids 265-354) GGRKRSETLSVHEASSIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK- CHFENTFFYKKLINGGYDYKSVREWTTKRKLG SEQ ID NO: 28 (MutC bdSENP1 amino acids 265-354) GGRKSSETLSVHEASAIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK- CHFENTFFYKKLINGGYDYKSVRGWTTVRKLG SEQ ID NO: 29 (MutD bdSENP1 amino acids 265-354) GGRKPSETLSVHEASEIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK- CHFFNTFFYKKLINGGYDYKSVREWTTQRKLG SEQ ID NO: 30 (MutE bdSENP1 amino acids 265-354) GGRKRSETLSVHEASGIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRYWTTARKLG SEQ ID NO: 31 (MutF bdSENP1 amino acids 265-354) GGRKPSETLSVHEASCIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFENTFFYKKLINGGYDYKSVRLWTTRRKLG SEQ ID NO: 32 (MutG bdSENP1 amino acids 265-354) GGRKSSETLSVHEASHIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRRWTTV-KLG SEQ ID NO: 33 (MutH bdSENP1 amino acids 265-354) GGRKPSETLSVHEASAIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLIN-GYDYKSVREWTTMRKLG SEQ ID NO: 34 (Muti bdSENP1 amino acids 265-354) GGRKKSETLSVHEASHIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFENTFFYKKLINGGYDYKSVREWTTRRKLG SEQ ID NO: 35 (Mut+ bdSENP1 amino acids 265-354) GGRKESETLSVHEASSIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRSWTTTRKLG SEQ ID NO: 36 (MutK bdSENP1 amino acids 265-354) GGRKVSETLSVHEASQIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFENTFFYKKLINGGYDYKSVRVWTTGRKLG SEQ ID NO: 37 (MutL bdSENP1 amino acids 265-354) GGRKLSETLSVHEASVIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFENTFFYKKLINGGYDYKSVRPWTTARKLG SEQ ID NO: 38 (MutM bdSENP1 amino acids 265-354) GGRKASETLSVHEASWIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRRWTTERKLG SEQ ID NO: 39 (MutN bdSENP1 amino acids 265-354) GGRKSSETLSVHEASPIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRRWTTRRKLG SEQ ID NO: 40 (MutO bdSENP1 amino acids 265-354) GGRKRSETLSVHEASRIVITREILQCLNDKEWLNDEVINLYLELLKERELREPNKFLK CHFFNTFFYKKLINGGYDYKSVRGWTTLRKLG SEQ ID NO: 41 (Mut1 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMK AIAFLFKGRRLRAERTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 42 (Mut2 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMT AIAFLFKGRRLRAECTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 43 (Mut3 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMH AIAFLFKGRRLRAEKTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 44 (Mut4 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMR AIAFLFRGRRLRAEVTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 45 (Mut5 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMT AIAFLFKGRRLRAEFTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 46 (Mut6 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMH AIAFLFKGRRLRAEQTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 47 (Mut7 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMD AIAFLFRGRRLRAECTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 48 (Mut8 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMP AIAFLFKGRRLRAEQTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 49 (Mut9 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMA AIAFLFKGRRLRAEYTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 50 (Mut10 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMT AIAFLFKGRRLRAERTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 51 (Mut11 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMS AIAFLFKGRRLRAEWTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 52 (Mut12 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMS AIAFLFKGRRLRAEHTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 53 (Mut13 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMS AIAFLFKGRRLRAEATPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 54 (Mut14 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMN AIAFLFKGRRLRAEWTPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 55 (Mut15 bdSUMO residues 1-97) MSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNEVFFRIKRSTQLKKLMNAYCDRQSVDMN AIAFLFKGRRLRAEATPDELEMEDGDEIDAMLHQTGG SEQ ID NO: 56 (MutA bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKPSETLSVHEASGIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVREWTTPRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 57 (MutB bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKRSETLSVHEASSIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVREWTTKRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 58 (MutC bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKSSETLSVHEASAIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRGWTTVRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 59 (MutD bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKPSETLSVHEASEIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVREWTTQRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 60 (MutE bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKRSETLSVHEASGIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRYWTTARKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 61 (MutF bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKPSETLSVHEASCIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRLWTTRRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 62 (MutG bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKSSETLSVHEASHIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRRWTTVKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 63 (MutH bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKPSETLSVHEASAIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGYDYKSVREWTTMRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 64 (Muti bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKKSETLSVHEASHIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVREWTTRRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 65 (MutJ bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKESETLSVHEASSIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRSWTTTRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 66 (MutK bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKVSETLSVHEASQIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRVWTTGRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 67 (MutL bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKLSETLSVHEASVIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFENTFFYKKLINGGYDYKSVRPWTTARKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 68 (MutM bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKASETLSVHEASWIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRRWTTERKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 69 (MutN bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKSSETLSVHEASPIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRRWTTRRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDFYSRDMELVFGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 70 (MutO bdSENP1 residues 248-481) PFVPLTDEDEDNVRHALGGRKRSETLSVHEASRIVITREILQCLNDKEWLNDE VINLYLELLKERELREPNKFLKCHFFNTFFYKKLINGGYDYKSVRGWTTLRKLGYNLIDC DKIFVPIHKDVHWCLAVINIKEKKFQYLDSLGYMDMKALRILAKYLVDEVKDKSGKQIDV HAWKQEGVQNLPLQENGWDCGMFMLKYIDEYSRDMELVEGQKHMSYFRRRTAKEILDLKA G SEQ ID NO: 71 (MBP) AGTGTSKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATG DGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALS LIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENG KYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVN KDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGR QTVDEALKDAQTN

[0231] The first four residues are part of a short linker between SUMOs and MBP.

EXAMPLES

Example 1 Cleavage Efficiency bdSENP1.SUP.MutB .and Other Site Specific Proteases

[0232] So far, bdSENP1 and Ulp1 are the most active Ubl-specific proteases known (Frey & Görlich 2014a). Around 15-50 nM of these two proteases were required to efficiently cleave the cognate substrate for 1h at 0° C. (FIG. 6). Other commonly used Ubl-specific proteases (i.e. xlAtg4, xlSub2 and bdNEDP1) are from 15 to 150-fold less efficient than bdSENP1 and Ulp1. Here, we showed that bdSENP1.sup.MutB protease was only 5-fold less efficient as compared bdSENP1 or Ulp1. bdSENP1.sup.MutB protease is therefore a more attractive option to some wild type Ubl-specific proteases.

[0233] This example also shows that the bdSENP1.sup.MutB protease is around 1000-fold more efficient than the site-specific protease from the tobacco etch virus (TEV protease). For instance, only 200 nM of bdSENP1.sup.MutB protease were enough to cleave ≈95% of the cognate substrate (bdSUMOmut 1), while even 10 μM of TEV protease were not enough to cleave the same amount of fusion protein at the same conditions of incubation (FIG. 6). Although TEV protease is the most extensively used protease for tag removal, our data proves that bdSENP1.sup.MutB represents a more powerful tool for the same propose.

Materials and Methods

[0234] Cleavage reactions were carried out using cleavage buffer (45 mM Tris/HCl pH 7.5, 250 mM NaCl, 2 mM MgCl.sub.2, 250 mM sucrose, 10 mM DTT) in a total volume of 20 μl. Prior to the reaction, substrates and proteases were diluted with cleavage buffer to 2-fold of the concentration required for the reaction. Equal volumes of diluted substrate and proteases were mixed in order to start the reaction. For every reaction, 100 μM of each MBP-tagged substrate were incubated with various amounts of a given protease (from 10 nM to 10 μM) for 1h on ice. The cleavage reactions were stopped by adding 180 μl of SDS sample buffer (3% SDS, 125 mM Tris/HCl (pH 6.8), 50 mM DTT, 1M sucrose and Coomassie brilliant blue G250). A sample corresponding to 2.5 μg of the bdSUMO.sup.Mut1-tagged substrate was resolved by SDS-PAGE and further stained by Coomassie blue. Control samples without protease were included in the assays. The proteases tested were: bdSENP1 from B. distachyon, Ulp1 from S. cerevisiae, SUMOstar protease, bdSENP1.sup.MutB protease, bdNEDP1 from B. distachyon, xIATG4B and xlSub2 from X. leavis and TEV protease.

Example 2 bdSENP1.SUP.MutB .and SUMOstar are Orthogonal Proteases

[0235] To test the orthogonality between the SUMOvera and SUMOstar systems, bdSUMO.sup.Mut1- and SUMOstar-MBP fusions were incubated for 1h at 0° C. with increasing concentrations of either bdSENP1.sup.MutB or SUMOstar protease (FIG. 7). On one hand, bdSENP1.sup.MutB protease only cleaved its cognate substrate at a concentration of 200 nM, whereas the SUMOstar-MBP fusion remained intact even at the highest protease concentration of 10 μM. On the other hand, SUMOstar protease only recognized SUMOstar-MBP and left intact the bdSUMO.sup.Mut1-MBP fusion protein even at the highest SUMOstar protease concentration of 10 μM. Note that 10 μM of either protease represents up to 1000-fold more protease needed for complete cleavage of the cognate substrate, and even so no protease cross-reactivity was observed. Therefore, the SUMOvera and the SUMOstar systems have indeed truly orthogonal specificities.

Materials and Methods

[0236] Cleavage reactions were performed, stopped and analyzed as described in Example 1. The only exceptions are that bdSUMO.sup.Mut1 and SUMOstar fusion proteins were incubated with ins creasing concentration of each protease.

Example 3 Expression of bdSUMO.SUP.Mut1.-Fusion Proteins in S. cerevisiae

[0237] Virtually, any protein tagged with a wild type SUMO protein is immediately cleaved if expressed in any eukaryotic host. SUMOstar is so far the only example of a SUMO protein that is a stable tag in different eukaryotic systems (Liu et al. 2008; Peroutka et al. 2008). We tested whether bdSUMO.sup.Mut1 would also be resistant to cleavage by Ulp1 in vivo and therefore stable in yeast cells.

[0238] To this end, we over-expressed citrine tagged with scSUMO, SUMOstar, bdSUMO or bdSUMO.sup.Mut1 in S. cerevisiae to test if the fusion protein would remain as a full-length product. The stability of the fusion proteins was analyzed by western blot using an anti-citrine antibody (FIG. 8). As expected, scSUMO- and bdSUMO-tagged citrines were completely cleaved, whereas SUMOstar- and bdSUMO.sup.Mut1-tagged proteins remained intact even after 6h of over-expression. In fact, bdSUMO.sup.Mut1 was the most stable fusion tag during recombinant protein expression in yeast (even more than SUMOstar) since up to 2100% of the over-expressed bdSUMO.sup.M′.sup.t1-tagged protein remained as full-length and almost no cleaved citrine was detected. These results suggest that bdSUMO.sup.Mut1 represents even a better choice than SUMOstar as a SUMO fusion tag for expression in S. cerevisiae.

Materials and Methods

[0239] For the over-expression of the SUMO-citrine fusion proteins, the respective expression plasmids were transformed in S. cerevisiae strain SFY123 (MATa, ADE2, his3-11, 15 leu2-3, 112 LYS2, trp1-1, ura3 cant-100, H2B-CFP::Trp1) using the PEG/LiAc protocol described in (Gietz & Schiestl 2007). Next, a single transformed colony was picked to inoculate a starting preculture of SD-medium supplemented with 2% (w/v) of glucose. After overnight incubation at 30° C., cells were pelleted by centrifugation for 5 min at 2,000 rpm and further resuspended using fresh medium supplemented with 2% (w/v) of glucose and 2% (w/v) of raffinose. This last process was repeated twice. Centrifugation and subsequent resuspension of cells with fresh medium supplemented with 2% (w/v) of glucose and 2% (w/v) of raffinose were repeated twice. Resuspended cells were then used to inoculate 250 ml of SD-medium supplemented with 2% (w/v) of raffinose to an initial concentration of OD.sub.600≈0.2. The culture was incubated at 30° C. with shaking until exponential growth phase was reached (OD.sub.600≈0.8-1.0). Over-expression of the SUMO-citrine fusion proteins was induced by the addition of 2% (w/v) of galactose for 6h at 30° C. while shaking. After protein over-expression, yeast cells were pelleted by centrifugation for 10 min at 2,000 rpm and 25° C. and further resuspended in resuspension buffer (45 mM Tris/HCl pH 7.5, 250 mM NaCl, 20 mM imidazole, 5 mM DTT).

[0240] To analyze the stability of the SUMO-citrine proteins, the resuspended cells were used to generate lysates by TCA/NaOH extraction as described in (Zuk 1998). Cell lysates corresponding to 35 mOD of cells expressing the citrine fusion proteins were resolved by SDS-PAGE and analyzed by western blot. An anti-GFP primary antibody was used to detect the presence of cleaved citrine and/or the full-length SUMO-citrine fusion protein. A cell lysate lacking a SUMO-tagged citrine was used as negative control.

Example 4 bdSUMO.SUP.Mut1 .is a Stable Tag in Eukaryotic Cellular Extracts

[0241] We also analyzed the stability of scSUMO-, SUMOstar-, bdSUMO- and bdSUMO.sup.Mut1-MBP fusion proteins in different eukaryotic extracts. Each MBP fusion protein was incubated in a highly concentrated extract for 2h at 25° C. and further analyzed by western blot (FIG. 9). Proteins tagged with SUMOstar and bdSUMO.sup.Mut1 were not cleaved in all samples, whereas the scSUMO as well as the bdSUMO fusions proteins were cleaved to different extents. Samples including a “protease mix” (Ulp1, SUMOstar protease, bdSENP1 and bdSENP1.sup.MutB protease, 1 μM each) showed that there was not any inhibitory substance that could have prevented endogenous SUMO-specific proteins to cleave either the SUMOstar or the bdSUMO.sup.Mut1 fusion proteins. Therefore, these results confirm that bdSUMO.sup.Mut1 is also a suitable fusion tag that can be used in virtually any eukaryotic host.

Materials and Methods

[0242] The stability of the different SUMO-tagged MBP fusion proteins was tested in different eukaryotic extracts (wheat germ extract, Xenopus laevis egg extract, rabbit reticulocytes extract, HeLa cell extract and Drosophila S2 cell extract). The preparation of the lysates was performed as described in (Mureev et al. 2009; Kovtun et al. 2010; Blow & Laskey 1986; Crevel & Cotterill 1991; Endo et al. 2010; Jackson & Hunt 1983). For a 12.5 μl volume reaction, 1 μM of SUMO-tagged substrate was incubated with 10 μl of each lysate for 2 h at 25° C. in the presence and absence of a protease mix containing 1 μM of different SUMO-specific proteases (Ulp1, SUMOstar, bdSENP1 and bdSENP1.sup.MutB protease). Finally, the reaction was stopped by adding SDS sample buffer (3% SDS, 125 mM Tris/HCl, (pH 6.8), 50 mM DTT, 1 M sucrose and coomassie brilliant blue G250) to a final volume of 100 μl. The stability of the substrates was analyzed by western blot using an anti-MBP primary antibody.

Example 5 Purification of a Hetero-Dimeric Complex in Yeast

[0243] Two or more site-specific proteases are used to purify protein complexes with defined subunit stoichiometry (Frey & Görlich 2014b). Although this technology is straightforward and requires only of proteases with mutually exclusive substrate specificity, it has been so far apply in prokaryotic systems such as E cob: Here, we show that the SUMOvera system together with the SUMOstar system can be used to purify dimeric protein complexes that are expressed in S. cerevisiae. As proof of principle, we selected the high affinity hetero-dimeric complex composed of the anti-GFP nanobody (Nb) (Kirchhofer et al. 2010) and the GFP-like protein named citrine (Heikal et al. 2000). Nb was cloned as an H14-bdSUMO.sup.Mut1-fusion protein, whereas citrine was fused to an N-terminal ZZ-SUMOstar tag (FIG. 10-A).

[0244] After the co-expression of both proteins, we used two consecutive capture-and-realize chromatographic steps as described in (Frey & Görlich 2014b) for the purification of the Nb.Citrine complex (FIG. 10-B). For the first chromatographic step, the cellular lysate containing the complex (soluble material) was loaded onto a column containing a Ni.sup.2+ chelate matrix to immobilize the dimeric complex via the H14-bdSUMO.sup.Mut1-Nb fusion protein. The non-bound material is then removed from the column after washing the matrix (non-bound material). The elution of the protein complex was then achieved by on-column protein cleavage using bdSENP1.sup.MutB protease. Notably, untagged citrine was present in the eluted complex due to the partial cleavage ZZ-SUMOstar tag by endogenous Ulp1 as observed in (FIG. 8). After a second affinity chromatographic step, the stoichiometric protein complex was obtained since the surplus of untagged Nb from elute 1 as well as the untagged Citrine were removed from the protein preparation (non-bound material 2). The stoichiometric complex is obtained by a second on-column protein cleavage using SUMOstar protease. Finally, the use of bdSENP1.sup.MutB and the SUMOstar proteases allowed obtaining an untagged and a purer complex as both affinity tags and all contaminants remained bound to both affinity matrices (Post elution with imidazole and SDS).

Materials and Methods

[0245] The transformation of the Nb.Citrine complex was performed in S. cerevisiae strain SFY123 (MATa, ADE2, his3-11, 15 leu2-3, 112 LYS2, trp1-1, ura3, can1-100. One plasmid codified for the fusion protein H14-bdSUMO.sup.Mut1-Nb and the second for the ZZ-SUMOstar-Citrine protein. Expression was performed as described in Example 3. After expression, the yeast pellet was resuspended in resuspension buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 5 mM DTT) to a final OD.sub.500 of 20-50/ml. A cocktail of different protease inhibitors was added to the resuspended cells to a final concentration of 1×. The stock (500×) of protease inhibitors contained the following compounds: 5 mg/ml aprotinin, 5 mg/ml leupeptin, 2.5 mg/ml elastatinal, 2.5 mg/ml chymostatin and 0.5 mg/mil pepstatin A. Cells were snap-frozen in liquid nitrogen and immediately thawn in a hot water bath for 10-15 min. The cellular membrane was disrupted using glass beads and constant vortexing to generate a cell lysate. Cell debris and insoluble material was removed from the lysed cells by ultracentrifugation at 38,000 rpm and 4° C. for 1.5h.

[0246] The purification of binary protein complexes was performed as described in (Frey & Görlich 2014b). Briefly, the cleared yeast lysate was incubated with Ni.sup.2+ chelate beads for 1h at 4° C. Subsequently, beads were place in a column and contaminant proteins were removed by adding 2 column volume (CV) of resuspension buffer. The protein complex was then eluted by adding 1CV elution buffer containing 200 nM of bdSENP1.sup.MutB protease for 111 at 4° C. For the second purification step, the complex was immobilized via the ZZ-tag (tandem repeat of the Z domain from staphylococcal protein A) using silica beads coupled to an anti ZZ-domain affibody. The sample was incubated with 2 ml of anti Z-domain beads for 1h at 4° C. After incubation, beads were washed with 2CV of resuspension buffer. For protein elution, silica beads were incubated with resuspension buffer supplemented with 100 nM of SUMOstar protease for 1h at 4° C. After elution, samples were pooled and frozen in liquid nitrogen for storage at −80°. Protein samples were taken at different steps of the expression and purification of the complex and analyzed by SDS-PAGE.

Example 6. Over-Expression of bdSENP1.SUP.MutB .Protease in S. cerevisiae

[0247] SUMO-specific proteases are the most efficient proteases for the removal of affinity tag from the protein of choice (Frey & Görlich 2014a; Malakhov et al. 2004). Unfortunately, this process can be only performed in vitro as the use of any exogenous SUMO-specific protease in vivo would compromise the viability of any eukaryotic cell. Since the site-specific proteolysis in vivo and specially using SUMO-specific proteases is of high relevance, we decided to test whether over-expression of bdSENP1.sup.MutB protease could be achieved without affecting the viability of yeast cells. Yeast cells were transformed with a high-copy vector encoding for Ulp1, bdSENP1, SUMOstar protease or bdSENP1.sup.MutB protease (FIG. 11). As a negative control, we transformed cells with a plasmid codifying for a bdSENP1 mutant (C440S) that is completely inactive, and therefore is not able to hydrolyze scSUMO. All transformed cells were able to grow when the expression of the proteases was repressed by the presence of glucose. After induction protease over-expression by the addition of galactose for 72h at 30° C., only yeast cells expressing either bdSENP1.sup.MutB protease or bdSENP1 (C440S) grew to a very similar. In contrast, over-expression of Ulp1, bdSENP1 and SUMOstar protease led to complete cellular death in the presence of galactose. bdSENP1.sup.MutB protease is therefore the only SUMO-specific protease that could be use to perform site-directed proteolysis in living yeast cells.

Materials and Methods

[0248] S. cerevisiae cells strain SFY123 (MATa, ADE2, his3-11, 15 leu2-3, 112 LYS2, trp1-1, ura3, can1-100) were used to test their viability after over-expression of a SUMO-specific protease. First, cells were transformed with a galactose inducible expression plasmid using the protocol described in (Gietz & Schiestl 2007). Transformed cells were inoculated in SD-medium supplemented with 2% (w/v) of glucose and further inoculated for 16h at 30° C. Cells were then pelleted and resuspended using fresh SD-medium supplemented with 2% (w/v) glucose and 2% (w/v) raffinose. Resuspended cells were incubated in of SD-medium containing 2% (w/v) of raffinose until exponential growth phase was reached (OD.sub.600≈1.0). Next, cells were sequentially diluted in 10-fold steps and 5 μl of each dilution were spotted in plates containing either galactose (0.02% and 0.2%) or glucose (2%). Plates were incubated for 72h at 30° C. and further scanned.

TABLE-US-00003 TABLE 1 Cleavage efficiency of different bdSUMO mutants by SUMO-specific proteases. scSUMO hsSUMO2 bdSUMO mutants .sup.C Protease .sup.A, B wt wt wt D67K Mut1 Mut8 Mut10 Mut11 Mut12 Mut13 Mut14 Mut15 Ulp1 ++++ + ++++ resistant resistant resistant resistant resistant resistant resistant resistant resistant SUMOstar ++++ + ++++ resistant resistant resistant resistant resistant resistant resistant resistant resistant protease hsSENP2 ++++ ++++ ++ resistant resistant resistant resistant resistant resistant resistant resistant resistant bdSENP1 ++ ++ ++++ + + + + + + ++ + + bdSENP1.sup.MutB resistant resistant + +++ ++++ ++ ++++ ++++ ++++ ++++ ++++ ++++ bdSENP1.sup.MutG + ++++ ++ +++ ++++ + ++++ +++ ++ +++ ++ +++ bdSENP1.sup.MutH resistant resistant + +++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ bdSENP1.sup.Muti resistant resistant + + ++++ + ++ ++ ++ +++ ++ ++ bdSENP1.sup.MutJ + ++ ++ ++++ ++++ ++ ++++ ++++ ++ ++++ ++ ++++ bdSENP1.sup.MutK resistant resistant ++ ++++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ .sup.A The protease concentration used for the reaction is sufficient to completely cleave 100 μM of the cognate SUMO protein within 1 h at 25° C. .sup.B Mutations of the tested bdSENP1 variants are shown in FIG. 15 .sup.C Mutations of the tested bdSUMO variants are shown in FIG. 15 +: ≤25% cleavage of the SUMO fusion protein ++: ≤50% cleavage of the SUMO fusion protein +++: ≤75% cleavage of the SUMO fusion protein ++++: ~100% cleavage of the SUMO fusion protein

LIST OF REFERENCES

[0249] Amor-Mahjoub, M. et al., 2006. The effect of the hexahistidine-tag in the oligomerization of HSC70 constructs. Journal of Chromatography B. Analytical Technologies in the Biomedical and Life Sciences, 844(2), pp. 328-334. [0250] Bachmair, a, Finley, D. & Varshaysky, a, 1986. In vivo half-life of a protein is a function of its amino-terminal residue. Science (New York, N. Y.), 234(4773), pp. 179-186. [0251] BOHNSACK, M. T., 2004. Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. RNA, 10(2), pp. 185-191. [0252] Butt, T., Malakhova, 0. & Malakhov, M., 2010. Methods and compositions for enhanced protein expression and purification. U.S. Pat. No. 7,655,413. [0253] Butt, T. R. et al., 2005. SUMO fusion technology for difficult-to-express proteins. Protein Expression and Purification, 43(1), pp. 1-9. [0254] Chan, P. et al., 2011. Purification of Heterotrimeric G Protein Subunits by GST-Ric-8 Association: PRIMARY CHARACTERIZATION OF PURIFIED Golf. Journal of Biological Chemistry, 286(4), pp. 2625-2635. [0255] Chant, A. et al., 2005. Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site. Protein Expression and Purification, 39(2), pp. 152-159. [0256] Chen, X., Pham, E. & Truong, K., 2010. TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector. Protein Science, 19(12), pp. 2379-2388. [0257] Frey, S. & Görlich, D., 2014a. A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. Journal of Chromatography A, 1337, pp. 95-105. [0258] Frey, S. & Görlich, D., 2014b. Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. Journal of Chromatography A, 1337, pp. 106-115. [0259] Harder, B. et al., 2008. TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms. BioTechniques, 44(6), pp. 765-772. [0260] Harper, S. & Speicher, D. W., 2011. Purification of Proteins Fused to Glutathione S-Transferase. In Methods in molecular biology (Clifton, N. J.). pp. 259-280. [0261] Hendriks, I. A. & Vertegaal, A. C. O., 2016. A comprehensive compilation of SUMO proteomics. Nature reviews. Molecular cell biology, 17(9), pp. 581-95. [0262] Herrmann, J., Lerman, L. O. & Lerman, A., 2007. Ubiquitin and ubiquitin-like proteins in protein regulation. Circulation Research, 100(9), pp. 1276-1291. [0263] Himeno, H., Kurita, D. & Muto, A., 2014. TmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell. Frontiers in Genetics, 5(APR), pp. 1-13. [0264] Katzmann, D. J., Babst, M. & Emr, S. D., 2001. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell, 106(2), pp. 145-155. [0265] Keiler, K. C., 2008. Biology of trans-Translation. Annual Review of Microbiology, 62(1), pp. 133-151. [0266] Kerscher, O., Felberbaum, R. & Hochstrasser, M., 2006. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annual review of cell and developmental biblogy, 22, pp. 159-80. [0267] Khorasanizadeh, S., Peters, I. D. & Roder, H., 1996. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nature structural biology, 3(2), pp. 193-205. [0268] Kimple, M. E., Brill, A. L. & Pasker, R. L., 2013. Overview of affinity tags for protein purification. Current Protocols in Protein Science, (SUPPL. 73), pp. 608-616. [0269] Kosobokova, E. N., Skrypnik, K. A. & Kosorukov, V. S., 2016. Overview of fusion tags for recombinant proteins. Biochemistry (Moscow), 81(3), pp. 187-200. [0270] Kostelansky, M. S. et al., 2007. Molecular Architecture and Functional Model of the Complete Yeast ESCRT-I Heterotetramer. Cell, 129(3), pp. 485-498. [0271] Kuwata, T. & Nakamura, T., 2008. BCL11A is a SUMOylated protein and recruits SUMO-conjugation enzymes in its nuclear body. Genes to Cells, 13(9), pp. 931-940. [0272] Malakhov, M. P. et al., 2004. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. Journal of Structural and Functional Genomics, 5(1-2), pp. 75-86. [0273] Marblestone, J. G. et al., 2006. Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein science: a publication of the Protein Society, 15(1), pp. 182-9. [0274] McCoy, J. & La Ville, E., 1997. Expression and Purification of Thioredoxin Fusion Proteins. In Current Protocols in Protein Science. Hoboken, N. J., USA: John Wiley & Sons, Inc., p. 6.7.1-6.7.14. [0275] Pattenden, L. K. & Thomas, W. G., 2008. Amylose Affinity Chromatography of Maltose-Binding Protein. In Affinity Chromatography. Totowa, N. J.: Humana Press, pp. 169-190. [0276] Rao, R. N., Allen, N. E. & Hobbs, J. N., 1983. Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli. Genetic and Enzymatic Basis of Hygromycin B Resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy, 24(5), pp. 689-695. [0277] Reverter, D. & Lima, C. D., 2004. A basis for SUMO protease specificity provided by analysis of human Senp2 and a Senp2-SUMO complex. Structure, 12(8), pp. 1519-1531. [0278] Reverter, D. & Lima, C. D., 2006. Structural basis for SENP2 protease interactions with SUMO precursors and conjugated substrates. Nature Structural & Molecular Biology 13(12), pp. 1060-1068. [0279] Sato, M. & Toda, T., 2007. Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast. Nature, 447(7142), pp. 334-337. [0280] Shen, L. et al., 2006. SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nature Structural & Molecular Biology, 13(12), pp. 1069-1077. [0281] Suh-Lailam, B. B. & Hevel, J. M., 2009. Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs. Analytical Biochemistry, 387(1), pp. 130-132. [0282] Taxis, C. & Knop, M., 2012. TIPI: TEV Protease-Mediated Induction of Protein Instability. In Methods in Molecular Biology. pp. 611-626. [0283] Vertegaal, A. C. O. et al., 2004. A proteomic study of SUMO-2 target proteins. Journal of Biological Chemistry, 279(32), pp. 33791-33798. [0284] Wang, K. H. et al., 2008. Tuning the strength of a bacterial N-end rule degradation signal. Journal of Biological Chemistry, 283(36), pp. 24600-24607. [0285] Woestenenk, E. A. et al., 2004. His tag effect on solubility of human proteins produced in Escherichia coli: A comparison between four expression vectors. Journal of Structural and Functional Genomics, 5(3), pp. 217-229. [0286] Xu, Z. et al., 2006. Crystal structure of the SENP1 mutant C603S-SUMO complex reveals the hydrolytic mechanism of SUMO-specific protease. The Biochemical journal, 398(3), pp. 345-352. [0287] YAN, Y., ORCUTT, & STRICKLER, J. E., 2009. The use of SUMO as a fusion system for protein expression and purification. Chimica oggi, 27(6). [0288] Zuo, X., Li, S., et al., 2005. Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. Journal of Structural and Functional Genomics, 6(2-3), pp. 103-111. [0289] Zuo, X., Mattern, M. R., et al., 2005. Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expression and Purification, 42(1), pp. 100-110.