WO2015049230A1 - Ensemble orthogonal de protéases se clivant à des étiquettes pour la purification de protéines et de complexes protéiques stoechiométriques - Google Patents

Ensemble orthogonal de protéases se clivant à des étiquettes pour la purification de protéines et de complexes protéiques stoechiométriques Download PDF

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WO2015049230A1
WO2015049230A1 PCT/EP2014/070918 EP2014070918W WO2015049230A1 WO 2015049230 A1 WO2015049230 A1 WO 2015049230A1 EP 2014070918 W EP2014070918 W EP 2014070918W WO 2015049230 A1 WO2015049230 A1 WO 2015049230A1
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seq
protease
prs
subunit
amino acid
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Dirk Goerlich
Steffen Frey
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention belongs to the field of biotechnology. More specifically, a widely applicable strategy for purification of recombinant protein complexes with defined stoichiometry is introduced. Further described is an orthogonal set of highly efficient and specific proteases that can be used for this procedure.
  • tags can be removed from the target protein during the purification process and thereby allow production of target proteins lacking any unwanted extensions at their termini.
  • This step is often accomplished by site- specific proteases recognizing a unique, short and linear recognition motif that has been artificially introduced between the tag and the target protein.
  • site-specific proteases recognizing a unique, short and linear recognition motif that has been artificially introduced between the tag and the target protein.
  • commercial suppliers offer various proteases, e.g. Thrombin, Factor Xa, enterokinase, or the 3C proteases from Tobacco etch virus (TEV) or human rhinovirus (Young et al. (2012), supra; Arnau et al. (2006) Protein Expr Purif 48: 1 - 13).
  • proteases In practice, the application of these proteases is often hampered by inefficient cleavage, a requirement for elevated temperature during cleavage, pronounced preferences for certain amino acids in the P1 ' position (the position after the scissile bond) or a narrow optimum for buffer and/or salt conditions. Also, most of these proteases leave unwanted residues at the N-terminus of the target protein (Arnau et al. (2006), supra). In addition, the specificity of some commonly used proteases (e.g. thrombin) is rather low, which might lead to the degradation of sensitive target proteins.
  • thrombin some commonly used proteases
  • the S. cerevisiae SUMO protease Ulpl p cleaves SUMO-containing substrates also in its cellular context.
  • SUMO small ubiquitin-related modifier
  • acceptor proteins can be covalently attached to numerous acceptor proteins, whereby an isopeptide bond is formed between SUMO ' s carboxy terminus and a lysine ⁇ -amino group from the acceptor (Muller et al. (2001 ) Nat Rev Mol Cell Biol. 2: 202-210).
  • the SUMO pathway involves two scUlpl -mediated proteolytic events:
  • the enzyme removes a C-terminal extension from the scSUMO precursor protein, thereby creating the characteristic C-terminal Gly-Gly motif present in the mature scSUMO.
  • scUlpl cleaves isopeptide bonds between scSUMO and acceptor proteins, and thereby reverses scSUMO modifications.
  • scUlpl exhibits an extraordinary specificity as it recognizes not just a short peptide motif, but the folded SUMO domain including the C-terminal Gly-Gly motif (Mossessova (2000) Mol Cell. 5: 865-876).
  • scUlpl can in principle accept any amino acid in the P1 ' position after the scissile bond (Malakhov et al. (2004), supra). It is therefore suited to generate a wide variety of non-acetylated N-termini, and thus allows restoring the authentic N-terminus of most target proteins.
  • the human orthologue of scUlpl (hsSENPI ) has been described previously (Gong et al. (1999) J Biol Chem. 275(5): 3355-3359).
  • SUMO is just one representative of a larger group of paralogous eukaryotic modifiers that also includes ubiquitin (Ub), Atg8 and NEDD8 (Yeh et al. (2000) Gene 248: 1 -14; van der Veen et al. (2012) Annu Rev Biochem 81 : 323-357). These proteins not only share a common fold and a similar conjugation mechanism, but also, they are similarly processed and deconjugated by dedicated proteases (van der Veen et al. (2012), supra). While SUMO, ubiquitin and NEDD8 possess a characteristic double-glycin (GG) motif at their mature C-termini, Atg8 proteins feature the sequence Phe-Gly (FG) at the corresponding position.
  • GG double-glycin
  • affinity tags Proteolytic removal of affinity tags is commonly accomplished in solution after elution from the affinity resin. While allowing free access of the protease to its substrate, this procedure has the disadvantage that the affinity tag released from the target protein has to be removed in a consecutive purification step. This generally necessitates a buffer exchange (to remove the prior used eluent) and a "reverse purification" on the same type of affinity resin. During this reverse purification step, the tag and any non-cleaved fusion protein (still containing the tag) are re-bound to the affinity resin and thus removed from the processed, tag- free target protein that now remains in the non-bound fraction.
  • affinity tags An alternative to such post-elution removal of affinity tags is on-column cleavage.
  • the target protein is released from the affinity resin by directly treating the loaded resin with a specific tag-cleaving protease (Walker et al. (1994) Biotechnology 12: 601 -605; Dian et al. (2002) J Chromatogr B Analyt Technol Biomed Life Sci 769: 133-144).
  • a protein is often not a single polypeptide but a complex comprising two or even multiple subunits. Structural and functional characterization of such protein complexes thus critically relies on purification strategies that allow controlling the stoichiometry of subunits.
  • Provided functional subunits can be produced in the absence of their binding partners, protein complexes can be assembled from individually pre-purified subunits (Fig. 1A). Alternatively, multiple subunits can be expressed and assembled in situ within the same host cell (Fig. 1 B, Fig. 2). In both cases, the assembled complex needs to be separated from an excess of non-assembled subunits and partially assembled sub-complexes (Fig. 1 , Fig. 2). This can be a challenging task, especially if the interaction between the two partners is regulated e.g. by binding to nucleotides or competing binding partners or if additional inactive subunits are in the mixture.
  • WO 2002/090495 (EP 1 392 717), US 6,872,551 , US 7,910,364, and US 7,498,165 describes a rapidly cleavable SUMO fusion protein expression system for difficult to express proteins. More specifically, SUMO or SUMO fragments are used to stabilize a poly-amino acid of interest and to enhance the solubility of the expressed fusion protein, enabling correct refolding and conferring monomeric expression without any toxic effects on the host cell.
  • WO 2003/057174 (EP 1 470 236) describes the use of SUMO and SUMO hydrolases/proteases in purifying polypeptides in general, but remains silent on the purification of stoichiometric protein complexes, or the use of these tools for on-column cleavage in affinity chromatography.
  • Gagnon et al. (2007) Methods in Enzymology 425: 263-282 discloses purification of a multidomain protein complex in which the sub domains have different affinity tags, but which are not linked via protease sites to the sub domains.
  • the present disclosure now introduces a general straightforward strategy for purification of stoichiometric protein complexes that exploits the combined discriminative power of two or more affinity matrices and proteases (Fig. 1 B, Fig. 2). Briefly, by tagging individual subunits of a given protein complex with orthogonal affinity tags and orthogonal protease recognition sites, consecutive sequences of affinity capture and proteolytic release allow selecting for the presence of each tagged subunit individually. This strategy thus provides a streamlined purification scheme and a defined stoichiometry alongside with a product purity conforming the highest standards.
  • figures 1 B and 2 only show the purification of a binary complex, protein complexes with more than two subunits can be purified in an analogous manner.
  • proteases with orthogonal (i.e. mutually exclusive) specificities.
  • such proteases should in addition be exceedingly efficient even at low temperature (preferably 0-4°C) and within a wide range of buffers.
  • new proteases matching these criteria are characterized in detail: bdSENPI and bdNEDPI from Brachypodium distachyon (bd) and ssNEDPI from salmon (Salmo salar, ss) (table 1 ).
  • additional sets of orthogonal substrate/protease pairs such as scAtg8/scAtg4 and xlUb/xlUsp2 are provided, which may be advantageously applied in the new method for purification of stoichiometric protein complexes.
  • Table 1 Nomenclature of NEDD8- and SUMO-orthologues used in this disclosure along with their corresponding proteases.
  • n.a. not analyzed; sc: Saccharomyces cerevisiae; bd: Brachypodium distachyon; ss: Salmo salar.
  • bdSENPI Comparing the new proteases to both, yeast scUlpl and a stabilized variant of TEV protease, bdSENPI was found to be possibly one of the most efficient and versatile proteases characterized for tag removal so far. It even outperforms scUlpl in several aspects. In addition, it is shown that most here-characterized members of the four classes of SUMO-, NEDD8-, Atg8- and ubiquitin-specific proteases possess orthogonal specificities. The present disclosure further describes the successful application of the new proteases, as exemplified by bdSENPI and bdNEDPI , for the purification of a stoichiometric complex.
  • the present disclosure is directed to 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),
  • AT N-terminal affinity tag
  • PRS protease recognition site
  • 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
  • step 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
  • 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
  • a first protease having an amino acid sequence with at least 45% identity over the full length of SEQ ID NO: 2 (bdSENPI ),
  • said protease is capable of cleaving the PRS according to SEQ ID NO: 1 (bdSUMO) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 2; preferably wherein the protease comprises the amino acid sequence shown as amino acids 1 -224 in SEQ ID NO: 2 (bdSENP1 24 8-48i ); more preferably wherein the protease consists of the amino acid sequence shown as amino acids 1 -224 in SEQ ID NO: 2 (bdSENP1 248-48 i ).
  • a second protease having an amino acid sequence with at least 70% identity over the full length of SEQ ID NO: 11 (ssNEDPI ), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 8 (ssNEDD8) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 1 1 ; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 11 (ssNEDPI ); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 1 1 (ssNEDPI ).
  • a third protease is provided, said protease having an amino acid sequence with at least 35% identity over the full length of SEQ ID NO: 12 (bdNEDPI ), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 9 (bdNEDD8) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 12; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 12 (bdNEDPI ); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 12 (bdNEDPI ).
  • Still another protease having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 20 (xlUb) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 21 ; preferably wherein the protease comprises the amino acid sequence as shown in SEQ ID NO: 21 (xlUsp2); more preferably wherein the protease consists of the amino acid sequence as shown in SEQ ID NO: 21 (xlUsp2).
  • a nucleic acid molecule, encoding any one of the above proteases is also contemplated.
  • a kit of parts comprising at least two proteases selected from
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 15 (scAtg4), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 15, wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 14 (scAtg8) with at least 20% activity as compared to the parent protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 15 (scAtg4); and
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 (xlUsp2), or a protease derivative thereof having an amino acid sequence with at least 80% identity over the full length of
  • protease derivative is capable of cleaving the PRS according to SEQ ID NO: 20 (xlUb) with at least 20% activity as compared to the parent protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 21 (xlUsp2);
  • proteases selected from (i)-(iii), more preferably comprising two proteases selected from (i) and (ii), and most preferably comprising the first protease and the third protease as described above.
  • a protease as described above, or the kit of parts as described above in a method of purifying stoichiometric protein complexes comprising at least two subunits wherein said at least two subunits comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), and optionally a spacer between the AT and the PRS, and wherein the AT of each of said at least two subunits differs from each other so to allow specific affinity chromatography, and wherein the PRS of each of said at least two subunits is cleavable by a protease which is orthogonal to the PRS of the other subunit(s); preferably wherein the method is further defined as described above.
  • AT N-terminal affinity tag
  • PRS protease recognition site
  • the purification of binary complexes according to this scheme is detailed Fig. 1 and Fig. 2.
  • a method for purifying a stoichiometric protein complex composed of at least two subunits from a 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
  • impurities e.g. monomers of the second of said at least two subunits
  • 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
  • step a) 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
  • impurities e.g. monomers of the first of said at least two subunits
  • 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 PCS of said second subunit, and
  • the stoichiometric protein complex is composed of at least two subunits.
  • the stoichiometric protein complex may also be composed of three, four, five, six, seven, eight or nine subunits, which each differ from each other.
  • the protein complex comprises a third subunit
  • a third affinity chromatography step may be incorporate. Such a third affinity chromatography step will further improve the purity, and it makes sure that only those complexes are purified, which contain all three subunits.
  • said third subunit comprised in said mixture comprises an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), wherein the AT of said third subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said third subunit, and wherein the PRS of said third subunit is cleavable by a protease, which protease is orthogonal to the PRS of the other two subunits, further comprising after step b) and prior to optional step c) an additional step b') subjecting the eluate from step b) to an affinity chromatography selective for the AT of the third subunit, whereby (i) the protein complex binds to the affinity resin via the AT of the third subunit, and
  • impurities e.g. monomers
  • the protein complex is eluted from the column and the AT of the third 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 third subunit, preferably wherein the protein complex is eluted by on-column cleavage, and
  • the protein complex comprises a fourth subunit
  • said fourth subunit comprised in said mixture comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), wherein the AT of said fourth subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said fourth subunit, and wherein the PRS of said fourth subunit is cleavable by a protease which is orthogonal to the PRS of the other three subunits, further comprising after step b') and prior to optional step c) an additional step b") subjecting the eluate from step b') to an affinity chromatography selective for the AT of the fourth subunit, whereby
  • impurities e.g. monomers
  • the protein complex is eluted from the column and the AT of the fourth 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 fourth subunit, preferably wherein the protein complex is eluted by on- column cleavage, and
  • the protein complex comprises a fifth subunit
  • said fifth subunit comprised in said mixture comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), wherein the AT of said fifth subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said fifth subunit, and wherein the PRS of said fifth subunit is cleavable by a protease which is orthogonal to the PRS of the other four subunits, further comprising after step b") and prior to optional step c) an additional step b'") subjecting the eluate from step b") to an affinity chromatography selective for the AT of the fifth subunit, whereby (i) the protein complex binds to the affinity resin via the AT of the fifth subunit, and
  • impurities e.g. monomers
  • the protein complex is eluted from the column and the AT of the fifth 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 fifth subunit, preferably wherein the protein complex is eluted by on-column cleavage, and
  • the method comprises the additional step of c) removing the protease from the eluate originating from the last affinity chromatography step.
  • step c) may be an affinity chromatography, a size exclusion chromatography, or a precipitation step, as generally known in the art.
  • any method suitable for removing the protease from the eluate may be applied.
  • the protease from the eluate originating from the last affinity chromatography prior to step c) comprises an affinity tag, as further defined below, and 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.
  • Said affinity tag of the protease may be the same than one of the affinity tags used in the affinity chromatography steps a), b), b'), b") or b'"), but with the provisio that it differs from the affinity tag used in the directly preceding affinity chromatography step b), b'), b") or b'").
  • the affinity tag of the final protease may be a polyHis-tag, and step c) is a Ni 2+ -chelate affinity chromatography.
  • the protein complex is eluted in step a) (iii) or step b) (iii) by on-column cleavage. More preferably both step a) (iii) and step b) (iii) are on-column cleavage steps.
  • the method further comprises optional steps b') (iii), b") (iii) or b'"), any one of step b') (iii), b") (iii), and b'") (iii) may be independently an on column-cleavage step.
  • steps b') (iii), b") (iii), and b'") (iii) are on-column cleavage steps, and more preferably all of steps b') (iii), b") (iii), and b"') (iii) are on-column cleavage steps.
  • On-column cleavage offers several advantages. It not only makes purifications more time-efficient by avoiding any lengthy buffer exchange and reverse chromatography steps.
  • On-column cleavage also allows the target proteins to be specifically released from the resin under very mild conditions: As the elution buffer differs from the washing buffer only by a minute amount of protease, on-column cleavage bypasses more drastic elution conditions as high concentrations of competitor, significant alterations in the buffer composition or pH changes. Most importantly, however, on-column cleavage potentiates the efficiency of protein purifications by elegantly combining the specificities of the affinity resin and the protease: Only proteins containing the proper affinity tag and the proper protease recognition site will be bound and consecutively released from the resin.
  • stoichiometric protein complex is intended to mean that each complex is composed of the same molar ratio of the same subunits, and that each complex has a definite identical size as defined by the number of subunits forming the complex.
  • one subunit A may form a complex comprising, e.g., either a subunit B or a subunit C, in which case there will be a mixture of stoichiometric protein complexes comprising subunits AB and complexes comprising subunits AC.
  • a stoichiometric protein complex is to be distinguished from random protein aggregates, which are characterized by a random molar distribution, and which differ by its constituents.
  • the “mixture” may be any suitable starting material for the purification method, such as an aqueous buffered or non-buffered solution comprising the stoichiometric protein complex.
  • the “mixture” may be a lysate, a supernatant, a pre-purified lysate or a pre-purified supernatant, or mixtures thereof, e.g. a mixture of lysates, a mixture of supernatants, or a mixture of a lysate and a supernatant, and the like.
  • the mixture may originate from a mixture of lysates and/or supernatants and/or a pre-purified solution, each comprising at least one of the subunits; or the mixture may originate from a single lysate or supernatant or pre-purified solution comprising all subunits of the protein complex.
  • impurities may also encompass an undesired buffered solution or a saline, undesired proteins other than the subunits of the complex, cell debris, and possibly monomers of the respective subunits and/or degradation products of said complex. Accordingly, apart from removing such monomers and/or degradation products, the method of the invention may also be used for replacing the buffered solution or saline, or for removing an undesired compound within the buffered solution or saline.
  • a purification scheme employing three or more orthogonal tags and proteases can be used for a straightforward purification of stoichiometric triple or higher order complexes.
  • the method allows for the purification of complexes comprising each orthogonally tagged subunit at least once. More specifically, the method is ideally suited for the purification of stoichiometric complexes if each orthogonally tagged subunit is comprised in the complex exactly once. If the protein complex is composed of two subunits, it preferably has a stoichiometry of 1 :1 .
  • each of the subunits may be comprised once, twice or more often in the protein complex. For example, if the protein complex is composed of 2 different subunits, it may have a stoichiometry of 1 :1 , 1 :2, 2:1 , 2:2, 1 :3, 3:1 , 2:3, 3:2, or 3:3, etc.
  • additional affinity chromatography steps can be done for each subunit comprised in the stoichiometric protein complex, as long as there are enough orthogonal protease / PRS systems.
  • orthogonal is intended to mean that the protease exhibits only cleavage activity against its corresponding substrate recognition sequence, but not on the other PRS or sequences in the subunits.
  • one PRS comprises, preferably consists of
  • cleavage reactions are performed in LS-buffer (250mM NaCI, 40mM Tris/HCI pH7.5, 2mM MgCI 2 , 250mM sucrose, 2mM DTT, 2pg/ml BSA).
  • substrates and proteases are pre-diluted in LS-buffer to twice the aspired end- concentration.
  • Cleavage is initiated by mixing identical volumes of substrate and protease pre-dilutions and stopped by mixing with a 9-fold excess of hot SDS sample buffer. A fraction corresponding to 2.5pg of substrate is separated by SDS- PAGE on 7-15% gradient gels. Gels are stained with Coomassie G250 and scanned. Cleavage activity can then be determined using e.g. a densitometer.
  • the most efficient orthogonal protease is used in the final affinity chromatography step, in order to keep the protease "contamination" in the final product low.
  • the PRS as defined in (i) or (ii) above is preferably comprised in the "last" subunit to be selected for, e.g. if the stoichiometric protein complex comprises two subunits, said PRS is comprised in the second subunit. More preferably, the last subunit (e.g. the second subunit, in case the complex comprises two kinds of subunits) comprises a PRS consisting of SEQ ID NO: 1 (bdSUMO).
  • the AT of the subunit comprising the bdSUMO-PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence shown in amino acids 1-224 of SEQ ID NO: 2 (bdSENPI 248 - 4 8i ), or (ii) a protease derivative of (i) having an amino acid sequence with at least 45% identity, preferably with at least 50% identity, more preferably with at least 60% identity, even more preferably with at least 70% identity, still even more preferably with at least 80% identity, most preferably with at least 90% identity, and even most preferably with at least 95% identity, such as 98% identity over the full length of SEQ ID NO: 2,
  • said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 1 (bdSUMO) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i), under identical conditions.
  • the AT is cleaved from the subunit using (i) the protease shown in in amino acids 1-224 of SEQ ID NO: 2 (bdSENP1 248-4 8i )-
  • one PRS comprises, preferably consists of
  • protease shown in SEQ ID NO: 4 is capable of cleaving said PRS derivative with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 3, under identical conditions.
  • the AT of the subunit comprising the scSUMO-PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 4 (scUlpl ), or
  • a protease derivative of (i) having an amino acid sequence with at least 35% identity, preferably with at least 40% identity, more preferably with at least 50% identity, even more preferably with at least 60% identity, still even more preferably with at least 70% identity, most preferably with at least 80% identity, and even most preferably with at least 90% identity, such as 95% or 98% identity over the full length of SEQ ID NO: 4,
  • said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 3 (scSUMO) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i), under identical conditions.
  • scSUMO SEQ ID NO: 3
  • one PRS comprises, preferably consists of
  • protease shown in SEQ ID NO: 7 is capable of cleaving said PRS derivative with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 5 or 6, respectively, under identical conditions.
  • the AT of the subunit comprising the hsSUMOI a- or hsSUMO2-PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 7 (hsSENPI ), or
  • protease derivative of (i) having an amino acid sequence with at least 45% identity, preferably with at least 50% identity, more preferably with at least 60% identity, even more preferably with at least 70% identity, still even more preferably with at least 80% identity, most preferably with at least 90% identity, and even most preferably with at least 95% identity, such as 98% identity over the full length of SEQ ID NO: 7, wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 5 (hsSUMOI a) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i), under identical conditions.
  • One PRS may comprise, preferably consist of
  • protease shown in SEQ ID NO: 11 is capable of cleaving said PRS derivative with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to when using the corresponding parent PRS with the amino acid sequence of SEQ ID NO: 8, 9 or 10, respectively, under identical conditions.
  • the AT of the subunit comprising the ssNEDD8-, bdNEDD8- or hsNEDD8-PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), SEQ ID NO: 12 (bdNEDPI ), and SEQ ID NO: 13 (hsNEDPI ), or
  • protease derivative of (i) having an amino acid sequence with at least 70% identity, more preferably with at least 80% identity, even more preferably with at least 90% identity, still even more preferably with at least 95% identity, most preferably with at least 98% identity over the full length of SEQ ID NO: 11 (ssNEDPI ); or with at least 70% identity, more preferably with at least 80% identity, even more preferably with at least 90% identity, still even more preferably with at least 95% identity, most preferably with at least 98% identity over the full length of SEQ ID NO: 13 (hsNEDPI ); or with at least 35% identity, preferably with at least 40% identity, more preferably with at least 50% identity, even more preferably with at least 60% identity, still even more preferably with at least 70% identity, most preferably with at least 80% identity, and even most preferably with at least 90% identity, such as 95% or 98% identity over the full length of SEQ ID NO: 12 (bdNEDP1 ); wherein said protease derivative
  • the PRS comprising an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8), SEQ ID NO: 9 (bdNEDD8) or a PRS derivative thereof as defined in (ii) is comprised in the first subunit. More preferably, the first subunit comprises a PRS comprising an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8) or SEQ ID NO: 9 (bdNEDD8), in particular wherein the first subunit comprises a PRS comprising an amino acid sequence as shown in SEQ ID NO: 9 (bdNEDD8).
  • the first subunit comprises a PRS consisting of an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8) or SEQ ID NO: 9 (bdNEDD8). It is thus particularly preferred that the first subunit comprises a PRS consisting of an amino acid sequence as shown in SEQ ID NO: 9 (bdNEDD8).
  • the AT of the subunit comprising one of these preferred PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), and SEQ ID NO: 12 (bdNEDPI ), or
  • a protease derivative having an amino acid sequence with at least 70% identity, more preferably with at least 80% identity, even more preferably with at least 90% identity, still even more preferably with at least 95% identity, most preferably with at least 98% identity over the full length of SEQ ID NO: 11 (ssNEDPI ); or with at least 35% identity, preferably with at least 40% identity, more preferably with at least 50% identity, even more preferably with at least 60% identity, still even more preferably with at least 70% identity, most preferably with at least 80% identity, and even most preferably with at least 90% identity, such as 95% or 98% identity over the full length of SEQ ID NO: 12 (bdNEDPI );
  • said protease derivative cleaves the PRS according to SEQ ID NO: 9 (bdNEDD8) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i), under identical conditions.
  • the AT of the subunit comprising the ssNEDD8 or bdNEDD8-PRS is cleaved off using a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), and SEQ ID NO: 12 (bdNEDPI ).
  • the AT of the subunit is cleaved off using the protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 12 (bdNEDPI ).
  • one PRS may comprise, preferably consist of
  • the AT of the subunit comprising such an xlUb-derived PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 21 (xlUsp2), or
  • protease derivative of (i) having an amino acid sequence with at least 80% identity, preferably at least 90% identity, more preferably 95% identity, and most preferably 98% identity over the full length of SEQ ID NO: 21 , wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 20 (xlUb) with at least 20%, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i).
  • one PRS comprises, preferably consists of
  • protease shown in SEQ ID NO: 15 is capable of cleaving said PRS derivative with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to when using the parent PRS with the amino acid sequence of SEQ ID NO: 14 under identical conditions.
  • the AT of the subunit comprising such a scAtg8-derived PRS is cleaved off using
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 15 (scAtg4), or
  • protease derivative of (i) having an amino acid sequence with at least 80% identity, more preferably with at least 90% identity, even more preferably with at least 95% identity, and most preferably with at least 98% identity over the full length of SEQ ID NO: 15, wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 14 (scAtg8) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease as defined in (i).
  • Atg4/Atg8 system in general is already described in the art (Li et al. JBC (201 1 ) 286(9): 7327-7338).
  • one PRS may comprise, preferably consist of the TEV protease recognition site shown in SEQ ID NO: 16 and 17.
  • the AT of the subunit comprising such an TEV-PRS is cleaved off using a TEV protease as shown in SEQ ID NO: 18 or a derivative thereof having an amino acid sequence with at least 80% identity, more preferably with at least 90% identity, even more preferably with at least 95% identity, and most preferably with at least 98% identity over the full length of SEQ ID NO: 18, wherein said protease derivative is capable of cleaving the TEV-PRS shown in SEQ ID NO: 16 with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the protease as shown in SEQ ID NO: 18.
  • a derivative is the protease as shown in SEQ ID NO: 19.
  • orthogonal protease systems are also likely to work, such as PreScission protease, enterokinase, Factor Xa, intein systems, and the like, if a subunit contains the respective PRS.
  • an amino acid sequence is said to have "X % sequence identity with SEQ ID NO: Y" over a defined length of amino acids if the sequence in question is aligned with said SEQ ID NO: Y and the sequence identity between those to aligned sequences is at least X%.
  • Such an alignment can be performed using for example publicly available computer homology programs such as the "BLAST" program, such as "blastp” provided at the NCBI homepage at http://www.ncbi.nlm.nih.gov/blast/blast.cgi, using the default settings provided therein.
  • the nature of amino acid residue changes by which the polypeptide having at least X% identity to a reference sequence differs from said reference sequence is a semi-conservative and more preferably a conservative amino acid residue exchange.
  • the subunit(s) may further comprise a spacer between the AT and the PRS, and/or between the PRS and the subunit.
  • the subunit(s) further comprise a spacer between the AT and the PRS.
  • a typical spacer should be flexible and hydrophilic, without representing a substrate for endogenous proteases or comprising a PRS as defined herein.
  • spacers having a high content of glycine and serine (as well as threonine and asparagine) are used.
  • charged residues especially negative charged residues are not excluded. The skilled person will recognize suitable spacers.
  • the affinity tag may be any affinity tag suitable in the above-described method.
  • any affinity tag may be used as long as it enables purification by affinity chromatography and as long as it is specific and does not interact with other affinity resins used in the method.
  • the AT may be a peptide tag, a covalent tag or a protein tag.
  • Examples of a peptide tag are an Avi-tag, a CBP (calmodulin-binding peptide)-tag, a Flag-tag, a HA-tag, a polyHis- tag, a Myc-tag, a S-tag, a SBP-tag, a Softag 1 , a Softag 3, a V5-tag, a Strep-tag or a Xpress-tag.
  • Examples of a covalent tag are Isopeptag and Spytag.
  • Examples for a protein tag are BCCP, GST-tag, GFP-tag, MBP-tag, NusA-tag, GFP-tag or a thioredoxin-tag.
  • the AT may be selected from the group consisting of a polyHis- tag, ZZ-tag, FLAG-tag, HA-tag, GST-tag, GST-epitope tag, GFP-tag, thioredoxin, epitope tag of thioredoxin, Avi-tag, or another peptide tag.
  • the AT is selected from a polyHis-tag, ZZ tag, FLAG tag, HA tag, and GST tag; more preferably the AT is selected from a polyHis-tag and a ZZ-tag.
  • a resin that allows for a quick and highly efficient capture of target complexes is preferred.
  • bdNEDPI is ideally suited as the slightly higher amount of protease needed for efficient cleavage (in comparison to bdSENPI ) can be efficiently removed during the following purification step.
  • the first subunit comprises a polyHis-tag
  • the second subunit comprises a ZZ-tag
  • a protease featuring the highest possible specific activity as any added protease either has to be removed in an additional step or will remain in the final protein preparation as a contaminant. Therefore, it is recommended to use bdSENPI at this step. Further, a set of bdSENPI variants harboring different affinity tags are contemplated that can be used for efficient removal of the protease after on-column cleavage.
  • the first subunit comprises a NEDD8-PRS or NEDD8-PRS derivative as defined above, preferably the bdNEDD8-PRS
  • the second subunit comprises a SUMO-PRS or SUMO-PRS derivative as defined above, preferably the bdSUMO-PRS.
  • the following setup is chosen: polyHis-bdNEDD8-subunit1 and ZZ-bdSUMO-subunit2. The AT is then cleaved off using the corresponding protease, as defined for the PRS/protease systems above.
  • proteases itself as described in the following can be of great benefit for purifying stoichiometric protein complexes. Accordingly, provided is a protease having an amino acid sequence with at least 45% identity, preferably with at least 50% identity, more preferably with at least 60% identity, even more preferably with at least 70% identity, still even more preferably with at least 80% identity, most preferably with at least 90% identity, and even most preferably with at least 95% identity, such as 98% identity over the full length of SEQ ID NO: 2 (bdSENPI ), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 1 (bdSUMO) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% activity as compared to the parent protease with the amino acid sequence of
  • the protease comprises the amino acid sequence shown as amino acids 1-224 in SEQ ID NO: 2 (bdSENP1 24 e- 481 ). More preferably, the protease consists of the amino acid sequence shown as amino acids 1-224 in SEQ ID NO: 2 (bdSENP1 24 8-48i )-
  • protease having an amino acid sequence with at least 70% identity, preferably with at least 80% identity, more preferably with at least 90% identity, most preferably with at least 95% identity, and even most preferably with at least 98% identity over the full length of SEQ ID NO: 11 (ssNEDPI ), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 8 (ssNEDD8) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% as compared to the parent protease with the amino acid sequence of SEQ ID NO: 11.
  • the protease comprises the amino acid sequence as shown in SEQ ID NO: 11 (ssNEDPI ). In a more preferred embodiment, the protease consists of the amino acid sequence as shown in SEQ ID NO: 11 (ssNEDPI ).
  • protease having an amino acid sequence with at least 35% identity, preferably with at least 40% identity, more preferably with at least 50% identity, even more preferably with at least 60% identity, still even more preferably with at least 70% identity, most preferably with at least 80% identity, and even most preferably with at least 90% identity, such as 95% or 98% identity over the full length of SEQ ID NO: 12 (bdNEDPI ), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 9 (bdNEDD8) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100% as compared to the parent protease with the amino acid sequence of SEQ ID NO: 12,
  • the protease comprises the amino acid sequence as shown in SEQ ID NO:
  • Still another protease having an amino acid sequence with at least 80% identity, preferably with at least 85% identity, more preferably with at least 90% identity, most preferably with at least 95% identity, and even most preferably with at least 98% identity over the full length of SEQ ID NO: 21 (xlUsp2), wherein said protease is capable of cleaving the PRS according to SEQ ID NO: 20 (xlUb) with at least 20% activity, preferably at least 30% activity, more preferably at least 40% activity, even more preferably at least 50% activity, still more preferably at least 60% activity, still even more preferably at least 70% activity, most preferably at least 80% activity, even most preferably at least 90% activity such as more than 100%, as compared to the parent protease with the amino acid sequence of SEQ ID NO: 21.
  • the protease comprises the amino acid sequence as shown in SEQ ID NO: 21 (xlUsp2); more preferably the protease consists of the amino acid sequence as shown in SEQ
  • proteases described above may further comprise an affinity tag, in particular if said protease is used as the "final" protease in the above-described method.
  • the affinity tag of the protease may be chosen among those described above.
  • the affinity tag is a polyHis-tag.
  • nucleic acid molecules are contemplated, which encode one of the proteases described and disclosed herein.
  • protease/substrate pairs that have potential as general tools for purification of recombinant proteins and protein complexes.
  • these proteases allow for an efficient substrate cleavage at 0°C.
  • six of the seven proteases fall into five groups with orthogonal substrate specificity: (i) scUlpl and bdSENPI , (ii) bdNEDPI (iii) scAtg4, (iv) xlUsp2 and (v) TEV protease.
  • the natural substrate of one protease group (as defined above) will therefore not be efficiently recognized by a protease from another group.
  • the ssNEDPI enzyme is special as it is strictly orthogonal to groups (i), (iii) and (v), but shows some degree of cross-reactivity on an ubiquitin- containing substrate (see Fig. 5).
  • NEDP1/NEDD8 pairs from Brachypodium and salmon behave similar in most assays using the standard P1 ' -Ala substrates. This is surprising, especially when considering the moderate degree of conservation between the corresponding NEDP1 enzymes (see Fig. 3). According to the available structure of the human NEDD8-NEDP1 complex (Shen et al. (2005) EMBO J 24: 1341 - 1351 ), the significant differences seen with respect to their P1 ' preferences (Fig. 12) can most probably be attributed to significant exchanges in protease residues contacting the substrate C-terminal of the scissile bond.
  • NEDP1 enzymes towards orthologous substrates can easily be explained by the striking conservation between NEDD8 proteins: From a total of only 12 amino acid exchanges between salmon and Brachypodium, only 5 are non-conservative (Fig. 3). The two exchanges present within the putative interface with the proteases do not seem to crucially influence the recognition by the protease.
  • the species promiscuity of NEDP1 enzymes has interesting practical implications: As a given NEDD8 substrate can be cleaved by both, bdNEDPI and ssNEDPI , the protease used for cleavage can be chosen freely.
  • ssNEDPI is remarkably insensitive towards high salt or a suboptimal residue in the substrate ' s P1 ' - position.
  • the salmon enzyme might thus be the protease of choice when cutting suboptimal substrates or cleaving at special buffer conditions.
  • the SUMO orthologues analyzed herein show a low degree of sequence conservation (Fig. 3).
  • yeast SUMO (Smt3p) has a high similarity to the human SUMO1 isoform (hsSUMOI )
  • the bdSUMO is more related to hsSUMO2.
  • the SUMO proteases from yeast, Brachypodium and human show a low degree of sequence conservation.
  • Structural alignments including structure predictions for the Brachypodium enzyme) (Armougom (2006) Nucleic Acids Res. 34: W604-8), however, indicate that all these enzymes adopt a similar three-dimensional structure.
  • the substrate » enzyme interfaces of the respective yeast and Brachypodium complexes differ in a significant number of residues that may easily account for the differences regarding cleavage efficiency (Fig. 10) and salt- or P1 ' - sensitivity (Fig. 1 1 , Fig. 12) that could be detected in our assays.
  • the two enzymes cleave their natural substrates better than substrates containing orthologous SUMO variants (Fig.
  • bdSENPI cleaves the corresponding Brachypodium substrate even >150- fold more efficiently than the substrate containing scSUMO (Fig. 6).
  • TEV protease recognition site (“TEV site") e.g. after the GST tag. TEV protease is thus often considered as the first choice for removing affinity tags from target proteins. While comparing the catalytic properties of a stabilized variant of TEV protease to proteases of the SENP1 and NEDP1 enzyme families, it turned out that TEV protease has major limitations that should be considered in practice.
  • the effective turnover rate of TEV protease is poor. Even at 25°C and at high substrate concentrations, each molecule of TEV protease can cleave only -150 substrate molecules per hour (Fig. 14). In addition, because of the high K M of the reaction (50-90 ⁇ ) (Kapust et al. (2002) Biochem Biophys Res Commun. 294: 949-955; Kapust et al. (2001 ) Protein Eng. 14: 993-1000; Parks et al. (1995) Virology 210: 194-201 )), this turnover rate can only be reached at exceedingly high substrate concentration (>100-200 ⁇ ).
  • TEV protease At lower substrate concentrations, the number of substrate molecules cleaved per protease drops significantly. Consequently, regardless of the concentration of substrate to be cleaved, roughly the same amount of protease is required. In practice, these properties have two major consequences. First, a complete cleavage by TEV protease is hard to achieve and generally requires long incubation times at elevated temperature (generally 16-30°C, as recommended by the commercial suppliers) or high enzyme concentrations. Second, any cleavage product will be contaminated with a rather high fraction of protease unless the substrate can be supplied in unreasonably high concentrations (>200 ⁇ ). For applications in an analytical or semi-preparative scale, the potential of TEV protease is therefore limited.
  • the new proteases characterized here are highly efficient tag- removing enzymes.
  • the substrate/protease ratio required for efficient cleavage remains rather constant even at low substrate concentrations. Therefore, especially bdSENPI , bdNEDPI and ssNEDPI are ideally suited for driving tag removal to completion.
  • the amount of protease used for cleavage can be lowered according to the substrate concentration. As a rule of thumb, at 0°C one molecule of bdSENPI will cleave roughly 2-4 substrate molecules per second, i.e.
  • NEDD8-specific enzymes have an approximately 10- fold lower turnover rate. Nevertheless, the two NEDP1 proteases can still digest an up to 1000-fold excess of substrate within one hour at 0°C.
  • the remaining "contaminant" protease that is used for cleavage can be neglected for the most common laboratory purposes.
  • the protease concentration used for cleavage can, however, be further drastically decreased if the cleavage reaction is performed at higher temperature or for a longer time. This is easily possible as the characterized SUMO-and NEDD8-specific proteases remain fully active even after over-night incubation at 37°C or 20°C, respectively.
  • a complete removal of the protease is possible using a protease variant harboring an engineered affinity tag. Together, these measures should allow for the removal of even trace amounts of protease.
  • bdSENPI the most active enzyme provided herein, bdSENPI , even outperforms its yeast orthologue in several aspects: At standard conditions (see e.g. Fig. 4 and Fig. 10A), bdSENPI has a 2-3-fold higher specific activity as compared to scUlpl . In addition, bdSENPI can efficiently cleave substrates in a wide range of salt conditions while the yeast counterpart significant loses activity at NaCI concentrations above 250mM (Fig. 1 1 ). This finding contrasts the relatively mild salt sensitivity (30% remaining activity at 1 M NaCI) reported for scUlpl in the literature (Malakhov et al. (2004), supra).
  • NEDP1 enzymes Similar to bdSENPI , also the two NEDP1 enzymes show an excellent tolerance to high salt conditions. These enzymes can therefore conveniently be used as tag- removing proteases in a variety of different buffers.
  • the inventors routinely elute the target proteins directly from Ni 2+ chelate columns using 30nM (untagged) bdSENPI within one hour at 4°C. In the vast majority of cases, an efficient release of the target protein is observed, generally yielding target protein concentrations between 100 and 300 ⁇ (up to 120 mg/ml), probably mostly limited by the binding capacity of the resin.
  • kit of parts comprising at least two proteases selected from
  • a bdSENPI -derived protease e.g. the bdSENPI protease, as defined above,
  • a ssNEDPI -derived protease and bdNEDPI derived protease e.g. the ssNEDPI protease or bdNEDPI protease, as defined above
  • TEV-derived protease e.g. the protease shown in SEQ ID NO: 18 or SEQ ID NO: 19, as defined above,
  • Atg4-derived protease e.g. the scAtg4 protease, as defined above, and
  • the kit may also comprise more than two proteases, such as three, four or five proteases, which are orthogonal to each other.
  • the kit may comprise at least two, such as three proteases selected from (i)-(iii). More preferably the kit comprises two proteases selected from (i)-(iii). Even more preferably, the kit comprises two proteases selected from (i) and (ii).
  • the kit comprises a bdNEDPI -derived protease and a bdSENPI -derived protease, as defined above, e.g. the bdNEDPI and bdSENPI protease disclosed herein.
  • at least one of the proteases comprised in the kit comprises an affinity tag, as further described above.
  • the orthogonal proteases disclosed herein as well as the kit comprising these orthogonal proteases can be advantageously used in a method of purifying stoichiometric protein complexes comprising at least two subunits, wherein said at least two subunits comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), and optionally a spacer between the AT and the PRS, and wherein the AT of each of said at least two subunits differs from each other so to allow specific affinity chromatography, and wherein the PRS of each of said at least two subunits is cleavable by a protease which is orthogonal to the PRS of the other subunit(s).
  • the method is further defined as described above.
  • the orthogonal proteases disclosed herein as well as the kit comprising these orthogonal proteases can be advantageously used for on-column cleavage in an affinity chromatography.
  • a generally applicable method for purifying stoichiometric protein complexes is provided. It is parallelizable and therefore suitable for automation.
  • the method requires a system of orthogonal proteases, which are also provided herein, which orthogonal proteases are capable of specifically cleaving affinity tags.
  • orthogonal proteases allows an almost free choice of the N- terminus of any subunit or target protein following the PRS, including the authentic N-terminus.
  • the newly provided orthogonal proteases are capable of specifically cleaving in a wide range of buffer conditions, and can be suitably used for on-column cleavage.
  • bdSENPI is highly specific, exhibits an extraordinary activity, even at 0 °C, which is higher than the specific activity of, e.g., scUlpl or TEV protease, has low P1 '-sensitivity, and shows a high salt tolerance. It demonstrated a higher species-specificity and merely moderate cross-reactivity with scUlpl .
  • the NEDD8- specific protease from Brachypodium distachyon (bdNEDPI ) has not been annotated or predicted as a protein, in particular not as a protease, and the NEDD8-specific protease from Salmo salar (ssNEDPI ) has not been biochemically characterized yet. Like bdSENPI both proteases are highly specific, show high specific activity even at 0-4°C, and exhibit a high salt tolerance and a low P1 '-sensitivity.
  • the invention describes a quintary orthogonal protease system comprising bdSENP1/scUlp1 -proteases, the bdNEDPI -protease, TEV protease, the scAtg4-protease and the xlUsp2-protease.
  • Figure 1 Comparison of methodologies used to purify stoichiometric binary protein complexes.
  • a binary complex (subunits T1 and T2) is pre-formed from purified individual components. Further chromatographic steps are required to remove surplus single subunits and binding-incompetent subunits.
  • the binary complex ⁇ 1 ⁇ 2 is separated from surplus subunit T1 by binding to affinity resin 2 specifically recognizing the tag fused to component T2 and similarly cleaved off with a component T2-specific protease.
  • the protease can be removed via an adequate affinity resin (I). Protein complexes with more than two subunits can be purified in analogously using an appropriate number of orthogonal affinity matrices and orthogonal protease systems.
  • Figure 3 Structure-based sequence alignment of SUMO- and NEDD8- orthologues and SUMO/NEDD8-specific proteases with their human orthologues. Relevant protein sequences were assembled from available EST and genomic sequence and aligned based on the results obtained form the Expresso server (see Example 1 ). Residue conservation at each position was classified as similar (°) or identical ( ⁇ ). Amino acids near the interface to the respective binding partner were highlighted in grey boxes. Residues directly involved in peptide bond hydrolysis are marked in bold. No structures were available for bdSUMObdSENPI , bdNEDD8 » bdNEDP1 and ssNEDD8 » ssNEDP1.
  • FIG. 4 Activity of tag-cleaving proteases.
  • A General design of protease substrates. All substrates contain an N-terminal polyHis-tag (Hisi 4 or Hisi 0 ), a protease recognition site (box left of the scissile bond) and the target protein MBP (maltose-binding protein; MBP). To ensure equivalent cleavage conditions, in SUMO-, NEDD8-, scAtg8 and xlUb-containing substrates the scissile bond is followed by identical sequences.
  • B Protease titration. Protease substrates (100 ⁇ ) sketched in (A) were incubated for 1 h at 0°C (left) or at 25°C (right) in the presence of the corresponding proteases.
  • Proteases were titrated down from 10 ⁇ to 1 nM. Reactions were stopped by dilution in hot SDS sample buffer. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are the non-cut (full length) proteins (fl) and the larger cleavage products (lcp). Bands of the molecular weight marker (M r ) correspond to 40kD, 50kD (more intense band) and 60kD (not always visible).
  • Figure 5 Cross-reactivity between various substrate/protease systems.
  • A 100 ⁇ of indicated substrates (100 ⁇ ) were incubated with 10 ⁇ of indicated proteases and protease fragments for 3h at 25°C.
  • the protease concentrations used are thus up to 10.000-fold higher than the concentrations required for efficient cleavage of their own substrates.
  • the SUMO-proteases, bdNEDPI , scAtg4, xlUsp2, and TEV protease represent five orthogonal groups of proteases.
  • the ssNEDPI enzyme shows some proteolytic activity on the xlUb-MBP substrate after 3h incubation and is therefore not strictly orthogonal to xlUspl under these conditions. Numbers in brackets refer to the amino acid numbers of full-length bdSUMO or full-length bdSENPI , respectively.
  • FIG. 6 The SUMO-specific proteases show a clear species preference for their respective SUMO substrates, but are not fully orthogonal to each other. 100 ⁇ of indicated substrates were cleaved at various conditions with either scUlpl or bdSENPI .
  • the grey bars in the upper left and lower right panels in each of A and B mark lanes with efficient digestion of cognate protease/substrate pairs; the bars in the lower left and upper right panels in each of A and B, highlight lanes showing efficient digestion of substrates by the orthologous protease.
  • A One hour incubation at 0°C with varying concentrations of protease.
  • a «40-fold higher concentration of bdSENPI is needed for efficient cleavage of scSUMO- MBP as compared to scUlpl .
  • efficient cleavage of bdSUMO-MBP requires «10-fold higher concentration of scUlpl as compared to bdSENPI .
  • B Time course at 0°C with fixed concentration (300nM) of protease.
  • bdSENPI needs >150-times longer than scUlpl for >95% cleavage of the orthologous yeast substrate.
  • Figure 7 bdSENPI , bdNEDPI , scAtg4 and xlUsp2 can be used for on-column cleavage.
  • A Schematic representation of substrates used for on-column cleavage experiments using bdSENPI and bdNEDPI .
  • B, C A Ni 2+ chelate resin was pre-loaded with similar amounts of His-i -bdSUMO- GFP and His 4 -bdNEDD8-mCherry. 50 ⁇ aliquots were treated with indicated concentrations bdSENPI (B) or bdNEDPI (C) for 1 hour at 0°C. Control incubations were performed with buffer or with buffer containing 400mM imidazole. Resins and eluates were photographed upon illumination at 366nm. GFP and mCherry in the eluate fractions were quantified via their absorption at 488nm and 585nm, respectively. Numbers below the eluate fractions show the quantification results.
  • Efficient on-column cleavage occurred with 20nM bdSENPI and 300nM bdNEDPI , respectively.
  • the cleavage was specific as even at a >30-fold higher protease concentration, no significant elution of the nonspecific target protein was evident.
  • E Schematic representation of substrates used for on-column cleavage experiments using scAtg4 and xlUsp2.
  • F G: Analogous to (B) and (C), on-column cleavage using scAtg4 or xlUsp2 was analyzed using a Ni 2+ chelate resin pre-loaded with Hisi 4 -scAtg8-mCherry and Hisi 4 -xlUb-GFP. Specific substrate release was observed after 1 h at 0°C using 6 ⁇ xlUsp2 and 10 ⁇ scAtg4, respectively.
  • FIG. 8 On-column cleavage using polyHis-tagged and non-tagged TEV protease.
  • a Ni 2+ chelate resin was separately loaded with His-n-TEV-GFP (A) or His 0 -ZZ-TEV-GFP (B).
  • IgG sepharose was loaded with His 0 -ZZ-TEV- GFP (C).
  • 50 ⁇ aliquots of loaded resins were treated with indicated concentrations of polyHis-tagged or non-tagged TEV protease for 1 hour at 25°C. Control incubations were performed with buffer or 500 mM imidazole. Resins and eluates were photographed upon illumination at 366 nm.
  • TEV protease For efficient elution within one hour, however, TEV protease needs to be applied in high concentrations (3-1 ⁇ ) even at 25°C. Preparative-scale purifications using TEV protease are therefore expensive and lead to significant protease contaminations of the final product.
  • Figure 9 Purification of a tag-free binary complex with 1 :1 stoichiometry.
  • Figure 10 Cleavage kinetics, temperature dependence and temperature stability of tag-cleaving proteases.
  • Figure 11 Salt sensitivity of tag-cleaving proteases.
  • A Substrates were incubated for one hour at 0°C with the corresponding proteases in buffer containing the indicated concentrations of NaCI. Strikingly, scUlpl and scAtg4 show a pronounced sensitivity to high NaCI concentrations.
  • B, C scSUMO- (B) or bdSUMO- (C) containing substrates were incubated at 0°C in the presence of 250mM (upper panels) or 1 M NaCI (lower panels) with 300nM of their corresponding protease. Samples were taken after various time points and analyzed by SDS-PAGE. Grey bars mark lanes with efficient digestion of cognate protease/substrate pairs.
  • FIG. 2A Schematic representation of protease substrates with different P1 ' residues. Substrates follow the general outline shown in Fig. 2A. To analyze the sensitivity for non-preferred amino acid residues C-terminal of the scissile bond (P1 ' position), this position was mutated to methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). For TEV substrates, the respective residues were inserted before the original glycine residue.
  • TEV substrates were incubated for 1 hour with the indicated concentrations of TEV protease. To allow for efficient cleavage, the cleavage was performed at 25°C. Note that, in comparison to the proteases in (A), TEV protease was used at a 100-fold higher concentration.
  • Figure 13 Preference of scAtg4 and xlUsp2 for residues in the P1 ' position.
  • A Schematic representation of the protease substrates used in (B) and (C).
  • scAtg4-specific substrates sketched in (A) were incubated for 1 hour at 0°C with various concentrations of scAtg4.
  • the scAtg4 protease shows only a mild sensitivity for residues in the P1 ' position. At 1 ⁇ concentration, the protease can cleave an 80-100-fold excess of most substrates. Similar to other proteases, scAtg4 does not accept substrates harboring a proline in the P1 ' position.
  • the substrate concentration was varied from 300 ⁇ to 3 ⁇ while keeping the protease concentration constant.
  • the fraction of substrate cleaved by the TEV protease remains rather constant.
  • the relative amount of cleaved substrate increases at lower substrate concentrations (i.e. at higher protease:substrate ratio).
  • Figure 15 Truncation analysis of the bdSUMO/bdSENPI -, bdNEDD8/bdNEDP1 -, and scAtg8/scAtg4 substrate/protease pairs.
  • Figure 16 Detailed cleavage analysis of selected bdSUMO and bdSENPI truncations. 100 ⁇ of indicated substrates were incubated with various concentrations of indicated bdSENPI fragments for 1 h at 0°C. The reaction was stopped by dilution in hot SDS sample buffer. Cleavage products were analyzed by SDS-PAGE and Coomassie staining. Shown are the full-length substrate protein (fl) and the larger cleavage product (lcp). The general substrate design followed the scheme depicted in Figure 4A. Numbers in brackets refer to the amino acid numbers of full-length bdSUMO.
  • Figure 17 Activity of full-length TEV(SH) protease and a TEV(SH) variant lacking the six C-terminal amino acids.
  • TEV substrate ZZ-TEV-MBP
  • TEV(SH)AC6 TEV(SH)AC6
  • fl non-cut (full length) proteins
  • lcp larger cleavage products
  • SEQ ID NO: 6 (hsSUMO2; Homo sapiens SUMO2, amino acids 17-93)
  • SEQ ID NO: 7 (hsSENP1 ; Homo sapiens SENP1 , amino acids 419-644)
  • SEQ ID NO: 8 (ssNEDD8; Salmo salar NEDD8)
  • SEQ ID NO: 1 (ssNEDPI ; Salmo salar NEDP1 ) MDPVVLSYQDSLLRRSDVALLEGPHWLNDQVIGFAFEYFAAELFKGLGEAAIFISP EVTQFIKCAACPEDLALFLEPLGLASRRWVFLAVNDNSIQTAGGSHWSLLLFLRD SGHFAHYDSQSGGNSLHARRIATKLEPFLGSGRKVPFVEEPCPLQQNSYDCGM YVICNAEALCERARVEGSPRLPVQTITPAYITQKRLEWCRLIQRLDRD
  • SEQ ID NO: 12 (bdNEDPI ; Brachypodium distachyon NEDP1 )
  • SEQ ID NO: 14 (scAtg8; Saccharomyces cerevisiae autophagy-related protein 8)
  • SEQ ID NO: 15 (scAtg4; Saccharomyces cerevisiae autophagy-related protein 4)
  • SEQ ID NO: 17 Spacer sequence in TEV protease recognition site-containing substrates used for P1 ' -sensitivity assays is underlined; cf. Fig. 12)
  • SEQ ID NO: 18 (TEV protease; Tobacco etch virus Nla protease)
  • SEQ ID NO: 20 (xlUb; Xenopus laevis ubiquitin)
  • SEQ ID NO: 21 (xlUsp2; Xenopus laevis ubiquitin-specific processing protease 2)
  • said mixture comprising said protein complex and monomers of said at least two subunits
  • 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),
  • 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
  • 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
  • step a) 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
  • 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
  • step a) (iii) and/or step b) (iii) the protein complex is eluted by on-column cleavage.
  • said third subunit comprised in said mixture comprises an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS),
  • the AT of said third subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said third subunit
  • PRS of said third subunit is cleavable by a protease, which protease is orthogonal to the PRS of the other subunits,
  • step b) further comprising after step b) and prior to optional step c) an additional step b') subjecting the eluate from step b) to an affinity chromatography selective for the AT of the third subunit, whereby
  • the protein complex is eluted from the column and the AT of the third 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 third subunit, preferably wherein the protein complex is eluted by on-column cleavage, and
  • said fourth subunit comprised in said mixture comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS),
  • the AT of said fourth subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said fourth subunit
  • PRS of said fourth subunit is cleavable by a protease which is orthogonal to the PRS of the other subunits, further comprising after step b') and prior to optional step c) an additional step
  • step b' subjecting the eluate from step b') to an affinity chromatography selective for the AT of the fourth subunit, whereby
  • the protein complex is eluted from the column and the AT of the fourth 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 fourth subunit, preferably wherein the protein complex is eluted by on-column cleavage, and
  • said fifth subunit comprised in said mixture comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS),
  • the AT of said fifth subunit differs from the AT of the other subunits and allows affinity chromatography being selective for the AT of said fifth subunit
  • PRS of said fifth subunit is cleavable by a protease which is orthogonal to the PRS of the other subunits
  • step b) further comprising after step b") and prior to optional step c) an additional step
  • the protein complex is eluted from the column and the AT of the fifth 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 fifth subunit, preferably wherein the protein complex is eluted by on-column cleavage, and optionally removing the cleaved off AT of the fifth subunit.
  • protease shown in SEQ ID NO: 2 (bdSENPI ) 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: 1 , under identical conditions;
  • the second subunit comprises a PRS comprising SEQ ID NO: 1 (bdSUMO);
  • the second subunit comprises a PRS consisting of SEQ ID NO: 1 (bdSUMO).
  • protease shown in SEQ ID NO: 4 (scUlpl ) 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: 3, under identical conditions.
  • protease shown in SEQ ID NO: 7 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: 5 or 6, respectively, under identical conditions.
  • one PRS comprises, preferably consists of
  • PRS comprising an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8), SEQ ID NO: 9 (bdNEDD8) or a PRS derivative thereof as defined in (ii) is comprised in the first subunit;
  • the first subunit comprises a PRS comprising an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8) or SEQ ID NO: 9 (bdNEDD8), in particular wherein the first subunit comprises a PRS comprising an amino acid sequence as shown in SEQ ID NO: 9 (bdNEDD8); most preferably wherein the first subunit comprises a PRS consisting of an amino acid sequence as shown in SEQ ID NO: 8 (ssNEDD8) or SEQ ID NO: 9 (bdNEDD8), in particular wherein the first subunit comprises a PRS consisting of an amino acid sequence as shown in SEQ ID NO: 9 (bdNEDD8).
  • protease shown in SEQ ID NO: 15 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: 14 under identical conditions.
  • At least one PRS comprises, preferably consists of the TEV protease recognition site shown in SEQ ID NO: 16. 13.
  • protease shown in SEQ ID NO: 21 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: 20 under identical conditions.
  • a protease comprising, preferably consisting of the amino acid sequence shown in amino acids 1 -224 of SEQ ID NO: 2 (bdSENP1 2 8- 48i ), or
  • protease derivative is capable of cleaving the PRS according to ID NO: 1 (bdSUMO) with at least 20% activity as compared to the parent protease as defined in (i), under identical conditions;
  • subunit is eluted from the column using (i) the protease shown in in amino acids 1 -224 of SEQ ID NO: 2 (bdSENP1 248-4 8i )-
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 4 (scUlpl ), or
  • protease derivative is capable of cleaving the PRS according to ID NO: 3 (scSUMO) with at least 20% activity as compared to the parent protease as defined in (i), under identical conditions.
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 7 (hsSENPI ), 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: 7,
  • protease derivative is capable of cleaving the PRS according to ID NO: 5 (hsSUMOI a) with at least 20% activity as compared to the parent protease as defined in (i), under identical conditions.
  • a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), SEQ ID NO: 12 (bdNEDPI ), and SEQ ID NO: 13 (hsNEDPI ), or
  • protease derivative cleaves the PRS according to SEQ ID NO: 9 (bdNEDD8) with at least 20% activity as compared to the parent protease as defined in (i), under identical conditions;
  • a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), and SEQ ID NO: 12 (bdNEDPI ), or
  • protease derivative having an amino acid sequence with at least 70% identity over the full length of SEQ ID NO: 11 (ssNEDPI ) or with at least 35% identity over the full length of SEQ ID NO: 12 (bdNEDPI ), wherein said protease derivative, cleaves the PRS according to ID NO: 9 (bdNEDD8) with at least 20% activity as compared to the parent protease as defined in (i), under identical conditions;
  • the subunit is eluted from the column using a protease comprising, preferably consisting of the amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NO: 11 (ssNEDPI ), and SEQ ID NO: 12 (bdNEDPI ); and most 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: 12 (bdNEDPI ).
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 15 (scAtg4), or
  • protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 15, wherein said protease derivative is capable of cleaving the PRS according to ID
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 18 or 19 (TEV protease), or
  • protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 18 or 19, wherein said protease derivative is capable of cleaving the PRS according to SEQ ID NO: 16 (TEV) with at least 20% activity as compared to the parent protease as defined in (i).
  • a protease comprising, preferably consisting of the amino acid sequence shown in SEQ ID NO: 21 (xlUsp2), or
  • protease derivative of (i) having an amino acid sequence with at least 80% identity over the full length of SEQ ID NO: 21 , wherein said protease derivative is capable of cleaving the PRS according to ID NO: 20 (xlUb) with at least 20% activity as compared to the parent protease as defined in (i).
  • any one of embodiments 1-20 wherein the protein complex is composed of 4 different subunits, preferably with a stoichiometry of 1 :1 :1 :1.
  • 24 The method of any one of embodiments 1 -23, wherein the mixture originates from a mixture of lysates and/or supernatants and/or a pre-purified solution, each comprising at least one of the subunits.
  • step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
  • step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
  • step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
  • step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
  • step c) is an affinity chromatography, a size exclusion chromatography, or a precipitation step.
  • step c) comprises an affinity tag, preferably as defined in embodiment 26, 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.
  • a protease having an amino acid sequence with at least 45% identity over the full length of SEQ ID NO: 2 (bdSENPI ), wherein said protease is capable of cleaving the PRS according to ID NO: 1 (bdSUMO) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 2;
  • protease comprises the amino acid sequence shown as amino acids 1 -224 in SEQ ID NO: 2 (bdSENP1 24 8-48i );
  • protease consists of the amino acid sequence shown as amino acids 1 -224 in SEQ ID NO: 2 (bdSENP1 248-48 i ).
  • a protease having an amino acid sequence with at least 70% identity over the full length of SEQ ID NO: 11 (ssNEDPI ), wherein said protease is capable of cleaving the PRS according to ID NO: 8 (ssNEDD8) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 1 ;
  • protease comprises the amino acid sequence as shown in SEQ ID NO: 11 (ssNEDPI );
  • protease consists of the amino acid sequence as shown in SEQ ID NO: 11 (ssNEDPI ).
  • a protease having an amino acid sequence with at least 35% identity over the full length of SEQ ID NO: 12 (bdNEDPI ),
  • protease is capable of cleaving the PRS according to ID NO: 9 (bdNEDD8) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 12;
  • protease comprises the amino acid sequence as shown in SEQ ID NO: 12 (bdNEDPI );
  • protease consists of the amino acid sequence as shown in SEQ ID NO: 12 (bdNEDPI ).
  • protease is capable of cleaving the PRS according to SEQ ID NO: 20 (xlUb) with at least 20% activity as compared to the parent protease with the amino acid sequence of SEQ ID NO: 21 ;
  • protease comprises the amino acid sequence as shown in SEQ ID NO: 21 (xlUsp2);
  • protease consists of the amino acid sequence as shown in SEQ ID NO: 21 (xlUsp2).
  • a kit of parts comprising at least two proteases selected from
  • proteases more preferably comprising two proteases selected from (i) and (ii), and most preferably comprising the protease according to embodiment 34 and the protease according to embodiment 36.
  • kit of parts of embodiment 39 wherein at least one of the proteases further comprises an affinity tag, preferably an affinity tag as defined in embodiment 26.
  • said at least two subunits comprise an N-terminal affinity tag (AT) separated from the subunit by a protease recognition site (PRS), and optionally a spacer between the AT and the PRS, and
  • AT N-terminal affinity tag
  • PRS protease recognition site
  • each of said at least two subunits is cleavable by a protease which is orthogonal to the PRS of the other subunit(s);
  • the present inventors searched for highly specific and efficient proteases with orthogonal specificity to the Saccharomyces cerevisiae (sc) scSUMO/scUlpl system. To this end, they followed two alternative approaches: First, assuming that a large evolutionary distance would be sufficient to generate an orthogonal system, the present inventors looked for clearly identifiable SUMO and scUlpl orthologues in organisms that diverged from S. cerevisiae early in evolution. In an alternative approach, the present inventors looked for paralogous substrate/protease pairs participating in parallel protein modification pathways within the same cell, which might thus be optimized by natural evolution for mutually exclusive specificities. Nevertheless, given the possibility that such parallel protease pathways might still have overlapping specificities, it could not a priori be assumed that such paralogous substrate/protease pairs would be orthogonal.
  • Figure 3 shows a structure-based sequence alignment of the respective protein sequence in comparison to their putative human orthologues. Folded protein domains were subjected to structure-based sequence alignments using Expresso (Armougom et al. (2006), supra) and the following templates: hsSUMOI : 2G4D_B (Xu et al. Biochem J. 398: 345-352); hsSUM02/bdSUMO: 2CKH_B (Shen et al. (2006) Biochem J. 397: 297-288); scSUMO: 1 EUV_B (Mossessova et al. (2000), supra); hsSENP1/bdSENP1 : 2CKH_A (Shen et al.
  • the catalytic domains of the Brachypodium and Salmon proteases were over- expressed in soluble form in E. coli and purified on a Ni 2+ chelate resin using an engineered polyHis-tag.
  • the catalytic domain of the reference protease scUlpl (Malakhov et al. (2004), supra) and a stabilized version of TEV protease (TEV(SH); van den Berg et al. (2006) J Biotechnol. 121 : 291 -298) lacking the C-terminal autoinhibitory peptide (Nunn and Djordijevic, 2005, supra) were purified accordingly. All proteases could be purified in large amounts and were highly active.
  • the activity of the C-terminally truncated TEV(SH) variant used in all further experiments was indistinguishable from the full-length parent TEV protease (Fig. 17).
  • proteases involved in processing of SUMO-like modifiers the inventors expressed and purified the S. cerevisiae Atg8-specific protease Atg4p (scAtg4; Kirisako et al. (2000) J Cell Biol. 151 (2): 263-276) and the catalytic domain of the Xenopus laevis (xl) ubiquitin-specific protease Usp2 (xlUsp2; cf. SEQ ID NO: 21 ).
  • a preferred truncated fragment of xlUsp2 is xlUsp2 43- 3 8 3, which was used in the examples herein.
  • untagged proteases were obtained by proteolytically removing the polyHis-tag. Design and expression of protease substrates for cleavage assays in solution
  • the sequence after the scissile bond was Gly-Thr (GT), in agreement with the natural and preferred TEV recognition sequence (Kapust et al. (2002), supra; Kostallas et al. (201 1 ) PLoS One 6: e16136).
  • GT Gly-Thr
  • a ZZ-tag of 14kDa was fused to the N-terminus of the protease recognition site in order to allow for an easy electrophoretic discrimination between full length and cleaved substrate. All substrate proteins were expressed in E.
  • the inventors chose to directly compare the properties of all six proteases recognizing substrates with ubiquitin-like fold (scUlpl , bdSENPI , bdNEDPI , ssNEDPI , scAtg4, xlUsp2) to the established TEV protease in a defined in-vitro system.
  • TEV protease based on the solubility-enhanced and autocleavage-resistant TEV(SH) variant (Berg and Berglund, 2006) lacking the C-terminal autoinhibitory peptide (Nunn and Djordijevic, 2005), that displayed a catalytic activity indistinguishable from the parent enzyme (Fig. 17).
  • the amino acid sequence of this protease is detailed in SEQ ID NO: 19.
  • LS-buffer 250mM NaCI, 40mM Tris/HCI pH7.5, 2mM MgCI 2 , 250mM sucrose, 2mM DTT, 2pg/ml BSA.
  • substrates and proteases were pre-diluted in LS-buffer to twice the aspired end-concentration.
  • Cleavage was initiated by mixing identical volumes of substrate and protease pre-dilutions. After incubation, the reactions were stopped by dilution in hot SDS sample buffer. A defined fraction (generally corresponding to 2.5pg of substrate) was separated by SDS-PAGE on 7-15% gradient gels. Gels were stained with Coomassie G250 and scanned.
  • TEV protease Sigma-Aldrich #T4455
  • TurboTEV Nacalai USA #NU0102
  • SUMO-specific proteases bdNEDPI, scAtg4, xlUsp2 and TEV protease represent five orthogonal groups of proteases.
  • the Brachypodium orthologue bdNEDPI When used at high concentrations it can, however, cleave a substrate containing xlUb, although with low efficiency.
  • the Brachypodium orthologue bdNEDPI does not show this cross- reactivity.
  • This example illustrates that a priori a prediction of orthogonality based on sequence distance or evolutionary distance is virtually impossible.
  • the differences in cross reactivity on the xlUb-containing substrate of bdNEDPI and ssNEDPI shows impressively that it is not possible to directly extrapolate from the specificity found for a protease from a given species to the specificity of a corresponding orthologue of another species.
  • yeast and Brachypodium SUMO proteases prefer their natural SUMO substrates, but are not fully orthogonal.
  • TEV protease Due to the lower activity of TEV protease, however, efficient on-column cleavage within one hour required much higher enzyme concentrations and incubation at 25°C. Similar to bdSENPI and bdNEDPI , also scAtg4 and xlUsp2 were tested for their applicability in on-column cleavage (Fig. 7E-G). Both proteases could specifically release their substrate proteins from the resin, efficient substrate cleavage, however, required higher protease concentrations as compared to cleavage in solution (compare to Fig. 4).
  • E. coli strain NEB Express New England Biolabs harboring expression plasmids for both proteins was grown at 25°C in 200ml TB medium with appropriate antibiotics to an OD 6 oo of 6. The culture was diluted in 600ml fresh medium containing antibiotics and 0.1 mM IPTG and further shaken at 18°C over night.
  • the resin was extensively washed with lysis buffer containing 15mM imidazole followed by lysis buffer containing 250mM sucrose. Bound protein complexes were specifically released by incubation with lysis buffer containing 250mM sucrose and 500nM bdNEDPI for 1 h at 4°C. 1 ml of the eluate fraction was incubated with 1 ml anti-ZZ-resin for 1 h at 4°C with and extensively washed with lysis buffer followed by buffer WB2 (100mM NaCI, 10mM Tris/HCI pH 7.5, 5mM DTT).
  • buffer WB2 100mM NaCI, 10mM Tris/HCI pH 7.5, 5mM DTT.
  • a highly pure and stoichiometric binary complex could be eluted by incubation with 30nM bdSENPI for 1 h at 4°C in buffer WB2. Most importantly, the two target proteins were cleaved off from their tags during the purification procedure, thereby yielding a non-tagged complex. The stoichiometric nature of the complex could be verified by gel filtration.
  • the Brachypodium SENP1 enzyme is the most efficient tag-cleaving protease tested.
  • the SUMO-, NEDD8- and Atg8-specific proteases are highly active between 0°C and 37°C.
  • the temperature dependence of protease activity was analyzed by incubating a fixed concentration of substrate proteins at various temperatures with a limiting amount of the respective proteases (Fig. 10B). As expected, the cleavage efficiency increased from 0°C to 37°C for all substrate/protease pairs. Also in this assay, bdSENPI performed better than its yeast orthologue and consistently showed a more efficient cleavage of its substrate at all temperatures. In a direct comparison of the two NEDP1 enzymes, the Brachypodium enzyme was more active than its salmon counterpart - at least between 0°C and 25°C and showed a similar temperature dependence as scAtg4. The activity of Usp2 greatly improved at 37°C.
  • TEV protease was more active at higher temperatures, it was in this assay at all temperatures at least 10-fold less efficient than any of the other proteases tested. Thus, while all SUMO- and NEDD8-specific proteases tested can be used for efficient tag removal at 0°C, TEV protease needs higher temperatures, more enzyme and/or significantly longer incubation times for similar results.
  • the yeast scUlpl and scAtg4 enzymes are sensitive to high-salt conditions.
  • protease activity was assayed in the presence of various NaCI concentrations ranging from 200mM to 1 M (Fig. 1 1 A).
  • TEV protease and ssNEDPI showed the highest salt tolerance and were largely insensitive to salt concentrations up to 1 M.
  • a moderate activity loss of «30% and « 50% with increasing salt was observed for bdNEDPI and xlUsp2, respectively.
  • the bdSENPI enzyme efficiently cleaved its substrate in a wide salt range between 200 and 750mM NaCI, while a slightly decreased activity was noticeable at 1 M NaCI.
  • yeast enzymes scUlpl and scAtg4 showed a striking salt sensitivity and significantly lost activity between 200mM and 1 M NaCI.
  • a detailed analysis of the cleavage kinetics of scUlpl and bdSENPI indicated that scUlpl cleaves its substrate «50 times slower in the presence of 1 M NaCI as compared to 250mM NaCI (Fig. 1 1 B). In comparison, the salt-induced kinetic inhibition was only »3-fold for bdSENPI (Fig. 1 1 C).
  • bdSENPI showed the highest cleavage efficiency of all proteases tested. Strikingly, the advantage of bdSENPI over its yeast orthologue, which can already be seen at moderate ionic strength, is even further increased at higher salt.
  • a number of proteases show sensitivity towards the residue following the scissile bond within the substrate ( ⁇ position; see e.g. Arnau et al. (2006) and Kapust et al. (2002), supra).
  • the residues C-terminally flanking the scissile bond may be regarded as a part of the recognition sequence, i.e. such proteases cleave within their recognition sequences.
  • xlUsp2 was remarkably promiscuous and processed its P1 ' -Ala, -Met, -Tyr, -Arg and -Glu substrates with virtually identical efficiency (Fig. 13C). As the only protease tested here, xlUsp2 could even process a P1 ' -Pro substrate - although with significantly reduced efficiency.
  • SUMO-, NEDD8-, Atg8- and ubiquitin-specific proteases show high turnover rates also at low substrate concentrations.
  • the amount of protease needed for efficient substrate cleavage strongly depends on the substrate concentration.
  • the rate of substrate conversion is limited only by the maximal turnover rate of the enzyme (substrate conversions per enzyme per time unit).
  • the effective turnover is limited by the availability of the substrate.
  • the number of processed substrate molecules per molecule of enzyme drops when lowering the substrate concentration.
  • a measure for the transition between these two regimes is the Michaelis-Menten constant (KM).
  • the turnover rate reaches half of its maximum when the substrate concentration equals K M .
  • the two kinetic parameters (maximal turnover rate and K M ) are characteristic for each enzyme/substrate pair and can be used to describe and predict the performance of an enzyme at different substrate concentrations.
  • Fig. 14 the effect of substrate concentration on the protease activity was analyzed in two slightly different setups.
  • Fig. 14A the concentrations of both, substrate and protease were reduced proportionally in order to maintain a constant substrate/protease ratio
  • Fig. 14B the substrate concentration was titrated down over two orders of magnitude while keeping the absolute protease concentration constant.
  • the standard substrates see Fig. 4A were used.
  • the two NEDD8-specific proteases as well as scAtg4 and xlUsp2 showed a similar general behavior with low K M , although the maximal turnover rate of these enzymes was significantly lower.
  • the substrate turnover per protease is further reduced at low substrate concentration: When titrating down the substrate at constant protease concentration the fraction of cleaved substrate increased only marginally (Fig. 14B). Along the same lines, reducing the concentration of both the substrate and the protease significantly impaired cleavage (Fig. 14A). These results are consistent with the rather high KM of the reaction that is reported in the literature (50-90 ⁇ ) (Kapust et al. (2002); Kapust et al. (2001 ); Parks et al. (1995), supra). Thus, complete substrate cleavage by TEV protease generally requires a high protease/substrate ratio. At low substrate concentration, the required ratio is even higher.
  • a detailed truncation analysis was performed for selected substrate/protease pairs (Fig. 15).
  • Fusion proteins consisting of a N-terminal maltose-binding protein (MBP), a protease recognition site (PRS; here bdSUMO, bdNEDD8 or scAtg8) and the respective protease (bdSENPI , bdNEDPI , or scAtg4) harboring truncations at defined positions (Fig. 15A) were expressed in E. coli.
  • an in vivo cleavage of the fusion protein after the PRS as analyzed by SDS-PAGE of whole cell lysates in SDS sample buffer is indicative for a decent functionality of both the protease recognition site and or the respective protease.
  • bdSUMO 8 3-97 and bdSENPI 288-477 are sufficient for a basal activity of the bdSUMO/bdSENPI system.
  • bdNEDD8 4- 75, bdNEDPI -13-219, scAtg8 2 9-n6, and scAtg4 9 i -3 88 are required for a basal activity in the bdNEDD8/bdNEDP1 or scAtg8/scAtg4 systems, respectively.
  • Proper cleavage and stability of the proteins can be expected when using larger fragments. According to Fig.
  • bdSENPI 242-481 and bdSENPI 248-481 showed virtually identical activity, deletion of five more amino acids from the N-terminus of bdSENPI lead to a significant decrease in proteolytic activity (compare left and middle column to the right column of panels).
  • bdSUMO 2 -97 and bdSUMO 2 i-97 could be cleaved with identical efficiency while bdSUMO 23- 9 7 is cleaved with reduced efficiency and cannot be cleaved to completion (compare upper two rows to lower row of panels).
  • a preferred minimal bdSUMO/bdSENPI system is represented by bdSUMO 2 i-97 and bdSENPI 2 8 - 8 i -
  • Sequence Database entry ACX1 1 191.1 sequence 36453 from US 7,569,389
  • Sequence Database entry C0H840 UniProt

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Abstract

La présente invention concerne le domaine de la biotechnologie. L'invention concerne plus particulièrement une stratégie couvrant une vaste plage d'applications pour la purification de complexes de protéines de recombinaison présentant une stœchiométrie définie. L'invention concerne en outre un ensemble orthogonal de protéases spécifiques et hautement efficaces à utiliser pour cette procédure.
PCT/EP2014/070918 2013-10-01 2014-09-30 Ensemble orthogonal de protéases se clivant à des étiquettes pour la purification de protéines et de complexes protéiques stoechiométriques WO2015049230A1 (fr)

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WO2016075143A3 (fr) * 2014-11-10 2016-07-21 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Élimination d'étiquettes présentes sur des protéines exprimées dans des hôtes procaryotes et eucaryotes
CN108144586A (zh) * 2016-12-05 2018-06-12 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2016075143A3 (fr) * 2014-11-10 2016-07-21 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Élimination d'étiquettes présentes sur des protéines exprimées dans des hôtes procaryotes et eucaryotes
US10378003B2 (en) 2014-11-10 2019-08-13 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Tag removal from proteins expressed in pro- and eukaryotic hosts
CN108144586A (zh) * 2016-12-05 2018-06-12 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法
CN108144586B (zh) * 2016-12-05 2020-07-17 中国水产科学研究院黄海水产研究所 对氨基苯甲脒仿生亲和配基的仿生亲和纯化方法

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