WO2011038219A1

WO2011038219A1 - Proteases having improved stability, solubility and activity

Info

Publication number: WO2011038219A1
Application number: PCT/US2010/050184
Authority: WO
Inventors: Brad Hook; Rachel Friedman Ohana; James Robert Hartnett; Michael R. Slater
Original assignee: Promega Corporation
Priority date: 2009-09-24
Filing date: 2010-09-24
Publication date: 2011-03-31

Abstract

Isolated active NIa proteases, such as from the tobacco etch virus, are modified by truncating the protease such that C-terminal amino acids of the protease are deleted. The isolated active mutant NIa proteases may show one or more of increased proteolytic activity, improved stability, less precipitation during storage, improved maintenance of activity after storage, or improved solubility. The isolated active NIa proteases may include the C-terminal deletion in combination with other modifications, such as point mutations. Compositions and kits containing the isolated mutant NIa proteases and methods of using the isolated mutant NIa proteases are described.

Description

PROTEASES HAVING IMPROVED STABILITY, SOLUBILITY AND ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/245,458, the entire content of which is hereby incorporated by reference in its entirety for all purposes.

INTRODUCTION

[0002] Potyviruses are viruses that infect plants and are classified in the picornaviral superfamily. Potyviruses encode proteases, including the NIa protease, which are involved during viral replication.

[0003] NIa protein proteases, such as the Tobacco Etch Virus (TEV) NIa protease, recognize a seven amino acid consensus sequence, Glu-X-X-Tyr-X-Gln/Xa, (SEQ ID NO: 20) where X can be various amino acid residues, and Xa is serine or glycine. The protease cleaves its substrate between the Gin and Gly/Ser residues.

[0004] Wild-type TEV protease is a 49 kDa NIa protease containing at least two sites at which self-cleavage may occur. The first is at the Vpg cleavage site between the Vpg domain and the catalytic domain which when cleaved yields the Vpg domain of about 22 kDa and the 27 kDa catalytic domain. The second is a self-cleavage site within the 27 kDa catalytic domain, at which cleavage occurs between the methionine at position 218 and the serine residue at position 219 of the catalytic domain. TEV protease is a Cys protease and may be inhibited by thiol reagents such as iodoacetamide.

SUMMARY OF THE INVENTION

[0005] An isolated active mutant NIa protease from tobacco etch virus (TEV) having the amino acid residues corresponding to amino acids from position 231 to position 242 of SEQ ID NO: 1 deleted is provided. In certain embodiments, between 12 and 22 amino acids are deleted from the C-terminus of the isolated NIa protease. Such deletions may encompass a deletion of 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, up to 22 amino acids. In certain embodiments, amino acids corresponding to position 224 to position 242 of SEQ ID NO: 1 are deleted. In certain embodiments, TEV NIa proteases truncated at the C-terminus have increased proteolytic activity compared with the corresponding wild-type protease, or a corresponding protease that does not have the deletion of amino acids at the C-terminus.

[0006] Polynucleotides encoding the mutant proteases described here in are also provided.

[0007] Compositions and kits containing the NIa proteases described herein are also provided. The compositions may include DTT, EDTA, glycerol, detergent or a combination thereof. Kits may include a resin having the capacity to bind a polypeptide tag, optionally a His- tag, HQ tag, GST tag, or HaloTag® (HTv7) (SEQ ID NO: 19).

[0008] Methods of purifying a polypeptide linked to a polypeptide tag sequence, such as a metal binding tag, a His-tag, a HQ tag, a GST tag, or a tag having at least 80% identity with SEQ ID NO: 19 are also described. The tag may be linked at the N-terminus of the protease and in some embodiments the tag is separated from the protease by a peptide or polypeptide linker sequence. The method includes the steps of binding the polypeptide tag sequence to a matrix or resin and cleaving the polypeptide from the polypeptide tag sequence using a NIa protease described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 is a photograph of a gel demonstrating the effects of mutations introduced in the tobacco etch virus (TEV) protease around the Vpg cleavage site. The arrow marks the junction between the Vpg and catalytic domains.

[0010] Fig 2A is a graph showing the relative protease activities of TEV 1-6. Truncations of TEV were created by removing amino acids from the C-terminus. The resulting HTv7-VPG- TEV fusions were expressed in E. coli. Lysates were created and tested for TEV activity using the Protease-Glo™ Assay (commercially available from Promega).

[0011] Figure 2B is a graph showing the relative protease activities of TEV27vl-6. Truncations of TEV were created by removing amino acids from the C-terminus. The resulting HTv7-TEV fusions were expressed in E. coli. Lysates were created and tested for TEV activity using the Protease-Glo™ Assay (commercially available from Promega).

[0012] Figure 3 is a table showing buffers used to test the stability HQ:TEV5 (SEQ ID NO: 17) after 7 days of storage at a variety of temperatures; 40°C, 22°C, 30°C, 37°C and 44°C. [0013] Figure 4 shows photographs of protein gels showing the amount of degradation of HQ:TEV5 proteases stored at 4°C, 22°C, 30°C, 37°C or 44 °C in the buffers shown in Figure 3.

[0014] Figure 5 shows photographs of protein gels showing the solubility of HaloTag®- TEV5 protease and HaloTag®-TEV3 protease.

DETAILED DESCRIPTION

[0015] The present invention provides, among other things, mutated NIa proteases having improved solubility, activity, reduced cleavage or self-cleavage between the Vpg and the catalytic domain, or any combination of these features, particularly after storage over a period of time, compared with the activity of a corresponding protease not containing the mutation. In one embodiment, the mutated proteases comprise an amino acid sequence in which a C-terminal portion of the protease is deleted. In one embodiment, the C-terminal deletion provides a modified NIa protease having superior properties compared with the corresponding wild-type protease, or a corresponding protease that does not have the terminal amino acids deleted. The isolated active NIa proteases may show one or more improvements such as increased proteolytic activity, improved stability, less precipitation during storage, increased activity after storage or increased solubility. Improvements in properties may be compared with a comparable NIa protease that does not contain the C-terminal deletion. The C-terminal deletion of the isolated active NIa protease may be combined with other modifications, such as point mutations.

[0016] Mutated NIa proteases, such as TEV protease, may be useful, for example, in one or more of protein purification and isolation, target directed proteolysis in vivo, and structural and functional mapping of a protein of interest in its native environment in vivo.

[0017] The potyviral wild-type NIa protease is about 49 kDa and is comprised of two domains: the Vpg domain (about 22 kDa) at the N-terminus and the catalytic domain (about 27 kDa) at the C-terminus. The Vpg and catalytic domains are separated by a NIa (Vpg) cleavage site. The NIa protease may be cleaved at the Vpg site to produce the Vpg domain (about 21.5 kDa) and the catalytic domain (about 27 kDa). Both the 49 kDa protease and 27 kDa catalytic domains have protease catalytic activity. The 27 kDa catalytic domain contains a non-canonical cleavage site between the methionine at position 218 and the serine residue at position 219 of the catalytic domain. NIa proteases comprising the C-terminal amino acid deletions described herein may show reduced self-cleavage at the corresponding non-canonical cleavage site between the serine at 218 and the Methionine at 219 of the corresponding catalytic domain. For example, isolated active mutant NIa protease may show reduced self-cleavage activity between amino acid residues corresponding to position 218 (Met) and 219 (Ser) of SEQ ID NO: 1 relative to a corresponding full-length protease.

[0018] NIa proteases, as used herein, are proteases that cleave the following peptide sequence Glu-Xaa¹-Xaa²-Tyr-Xaa³-GlnJ,Xaa⁴ (SEQ ID NO: 20) with cleavage indicated by the arrow, between the Gin and the Xaa⁴, where Xaa¹, Xaa² and Xaa³, which may be the same or different, represent an amino acid and Xaa⁴ represents Ser or Gly. NIa proteases include proteases that contain a full-length, partial or mutated Vpg domain in addition to a protease catalytic domain. NIa proteases also include proteases that include the protease catalytic domain but do not include a Vpg domain or a partial sequence of a Vpg domain. As used herein, activity of a NIa protease means the capacity of the protease to cleave a protein or polypeptide substrate containing the Glu-Xaa^Xaa^Tyr-Xaa^GlnjSer or Gly (E-X-X-Y-X-QjS/G ; SEQ ID NO: 20) at that recognition site. Examples of recognition sites include EDLVEQjS, EDLVEQjG, EIIYTQjS, EIIYTQjG, ETIYLQjS, ETIYLQjG, EPVYFQjS, EPVYFQjG, ELVYSQjS, ELVYSQjG, ENLYFQjS, and ENLYFQjG. Suitably, NIa proteases of the invention have an activity that is at least about 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 100%, 105% 110%, 115%, 120%, 125%, 130%, 140%, 150%, 175% or 200% of their corresponding wild-type NIa protease or of the wild-type 27 kDa TEV protease, or of the corresponding protease that does not contain the mutation.

[0019] The invention provides mutated NIa proteases that show improved stability over time relative to the corresponding wild-type NIa protease, or to the corresponding NIa protease that does not contain the mutation. For example, mutated NIa proteases may retain activity for a longer period after storage compared with their corresponding wild-type protease, with the wild- type 27 kDa TEV protease, or with the corresponding NIa protease that does not contain the mutation. For example, the improved stability is from about 25-99%, such as at least about 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99 % of the original protease activity after storage at -20°C, 4°C and/or 22°C for 1, 2, 3, 4, 8, 16, 20, 26, 39 or 52 weeks (or 1 month, 2 months, 3 months, 4 months, 6 months, 9 months or 12 months). For example, from about 25- 99%, for example, at least about 25, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99 % of the protease suitably remains soluble after storage at -20°C, 4°C and/or 22°C for 1, 2, 3, 4, 8, 16, 20, 26, 39 or 52 weeks (or 1 month, 2 months, 3 months, 4 months, 6 months, 9 months or 12 months).

[0020] Examples of NIa proteases may include those derived from or synthesized based on potyviruses such as the Alstroemeria mosaic virus, Amaranthus leaf mottle virus, Apium virus Y, Parsley virus Y, Araujia mosaic virus, Artichoke latent virus, Asparagus virus, Banana bract mosaic virus, Bean common mosaic virus, Azuki bean mosaic virus, Blackeye cowpea mosaic virus, Dendrobium mosaic virus, Peanut chlorotic ring mottle virus, Peanut mild mottle virus, Peanut stripe virus, Bean common mosaic necrosis virus, formerly serotype A of BCMV, Bean yellow mosaic virus, Croatian clover virus, Crocus tomasinianus virus, White lupin mosaic virus, Pea mosaic virus, Beet mosaic virus, Bidens mottle virus, Calanthe mild mosaic virus, Calanthe mosaic virus, Carnation vein mottle virus, Carrot thin leaf virus, Carrot virus Y, Celery mosaic virus, Ceratobium mosaic virus, Chilli veinal mottle virus, Pepper vein banding mosaic virus, Clitoria virus Y, Clover yellow vein virus, Pea necrosis virus, Statice virus Y, Cocksfoot streak virus, Colombian datura virus, Petunia flower mottle virus, Commelina mosaic virus, Cowpea aphid-borne mosaic virus, Sesame mosaic virus, South African passiflora virus, Cowpea green vein banding virus, Cypripedium virus Y, Dasheen mosaic virus, Datura shoestring virus, Diuris virus Y, Endive necrotic mosaic virus, Freesia mosaic virus, Gloriosa stripe mosaic virus, Groundnut eyespot virus, Guinea grass mosaic virus, Helenium virus Y, Henbane mosaic virus, Hibbertia virus Y, Hippeastrum mosaic virus, Hyacinth mosaic virus, Iris fulva mosaic virus, Iris mild mosaic virus, Iris severe mosaic virus, Bearded iris mosaic virus, Japanese yam mosaic virus, Johnsongrass mosaic virus, Kalanchoe mosaic virus, Konjac mosaic virus, Leek yellow stripe virus, Garlic virus, Lettuce mosaic virus, Lily mottle virus, Rembrandt tulip breaking virus, Lycoris mild mottle virus, Maize dwarf mosaic virus, Moroccan watermelon mosaic virus, Narcissus degeneration virus, Narcissus late season yellows virus, Jonquil mild mosaic virus, Narcissus yellow stripe virus, Nerine yellow stripe virus, Nothoscordum mosaic virus, Onion yellow dwarf virus, Ornithogalum mosaic virus, Ornithogalum virus, Papaya leaf distortion virus, Papaya ringspot virus, Watermelon mosaic virus, Parsnip mosaic virus, Passion fruit woodiness virus, Pea seed-borne mosaic virus, Peanut mottle virus, Pepper severe mosaic virus, Pepper veinal mottle virus, Pepper vellow mosaic virus, Peru tomato mosaic virus, Pleione virus Y, Plum pox virus, Pokeweed mosaic virus, Potato virus A, Potato virus V, Potato virus Y, Rhopalanthe virus Y, Sarcochilus virus Y, Scallion mosaic virus, Scallion mosaic virus China, Shallot yellow stripe virus, Welsh onion yellow stripe virus, Sorghum mosaic virus, Soybean mosaic virus, Sugarcane mosaic virus, Sunflower chlorotic mottle virus, Sweet potato feathery mottle virus, Sweet potato russet crack virus, Sweet potato A virus, Sweet potato chlorotic leafspot virus, Sweet potato internal cork virus, Sweet potato latent virus, Sweet potato mild speckling virus, Sweet potato mild speckeling virus Argentina, Sweet potato virus G, Telfairia mosaic virus, Tobacco etch virus, Tobacco vein banding mosaic virus, Tobacco vein mottling virus, Tropaeolum mosaic virus, Nasturtium mosaic virus, Tuberose mild mosaic virus, Tulip breaking virus, Tulip mosaic virus, Turnip mosaic virus, Tulip top breaking virus, Tulip chlorotic blotch virus, Watermelon leaf mottle virus, Watermelon mosaic virus, Vanilla necrosis virus, Watermelon mosaic virus, Wild potato mosaic virus, Wisteria vein mosaic virus, Yam mild mosaic virus, Yam mosaic virus, Dioscorea green banding virus, Zantedeschia mosaic virus, Zea mosaic virus, Zucchini yellow fleck virus, Zucchini yellow mosaic virus.

[0021] In one embodiment, the mutated NIa protease has a C-terminal deletion in the catalytic domain. For example, the amino acid residues corresponding to amino acids from position 219 to 242, 220 to 242, 221 to 242, 222 to 242, 223 to 242, 224 to 242, 225 to 242, 226 to 242, 227 to 242, 228 to 242, 229 to 242, 230 to 242, 231 to 242, 232 to 242, 233 to 242, 234 to 242, 235 to 242, 236 to 242, 237 to 242, 238 to 242, 239 to 242, 240 to 242, or 241 to 242 of SEQ ID NO: 1 (the wild-type 27 kDa catalytic domain) may be deleted.

[0022] The mutated NIa protease may further include one or more mutations in combination with the C-terminal deletion. For example, the sequence around the Vpg cleavage site may be mutated, suitably to reduce cleavage at the Vpg site which separates the Vpg domain from the catalytic domain. For example, the wild-type tobacco etch virus sequence at the Vpg site comprises the sequence EDLTFEjGES (SEQ ID NO: 21), with cleavage indicated by the arrow, between the E (Glu) and G (Gly) (positions 6 and 7 of SEQ ID NO: 21). Suitable mutations include, without limitation, replacing the Gly at position 7 of SEQ ID NO: 21 with another amino acid such as a Ser or Val, replacing the Glu (E) at position 6 of SEQ ID NO: 21 with another amino acid such as a Leu (L), replacing the Glu (E) at position 8 of SEQ ID NO: 21 with another amino acid such as an Asn (N), replacing the Glu (E) at position 1 of SEQ ID NO: 21 with another amino acid such as Thr or Leu, or any combination thereof. The NIa protease may contain other mutations, such as replacing one or more amino acids corresponding to positions 17, 68 and/or 77 of SEQ ID NO: 1 (wt 27) with another residue. Suitably the residue corresponding to position 17 of SEQ ID NO: 1 is replaced by a Ser, the residue corresponding to position 68 of SEQ ID NO: 1 is replaced by an Asp, the residue corresponding to position 77 of SEQ ID NO: 1 is replaced by a Val, or any combination thereof. Suitably, the amino acid corresponding to position 1 of SEQ ID NO: 1 is replaced with another amino acid, such as a valine or a serine and the amino acid corresponding to position 2 of SEQ ID NO: 1 replaced with another amino acid, optionally an asparagine, or any combination thereof. These mutations may be present singly or in any combination.

[0023] In certain embodiments, the isolated active mutant NIa protease comprises at least a portion of or the entire Vpg domain. For example, the isolated active mutant NIa protease includes six amino acid residues corresponding to position 183 to 188 of SEQ ID NO: 2 in which the residue corresponding to position 188 is replaced with another residue, such as leucine, the residue corresponding to position 183 is replaced with another residue, such as threonine or leucine, or any combination thereof.

[0024] Suitably, the mutated NIa proteases have at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a wild-type NIa protease, such as TEV protease, or other naturally occurring iso forms having the same or similar substrate cleavage activity, or to one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,. SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. Optionally, the mutated NIa proteases have at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90%) sequence identity, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 4-9 that comprise an additional two amino acids MV (methionine/valine) at the N-terminus.

[0025] The present invention further provides polypeptides comprising mutated NIa proteases that are fused to a heterologous polypeptide, fusion polypeptide, or carrier polypeptide. In one embodiment, the heterologous polypeptide, fusion polypeptide, or carrier polypeptide facilitates isolation of the protease. Suitably, the heterologous polypeptide is a peptide or polypeptide tag that has an affinity for, or binds to, a resin or matrix. One or more different heterologous polypeptides, fusion polypeptides, or carrier polypeptides may be linked per mutated NIa protease. In one embodiment, the heterologous polypeptide includes a His-tag, such as a sequence comprising 4, 5, 6, 7, 8, 9, 10, 11, 12 or more consecutive histidine (His) residues. In another embodiment, the heterologous polypeptide includes a metal-affinity tag such as a HQ tag, for example, a sequence of "HQ" residues that repeat 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In one embodiment, the heterologous polypeptide includes a glutathione S-transferase (GST) tag (position 1 to 654 of SEQ ID NO: 40). In another embodiment, the heterologous polypeptide includes SEQ ID NO: 19 (HaloTag^®), or a sequence having at least about 80, 85, 90, 95, 97, 98 or 99 % identity with SEQ ID NO: 19. The heterologous polypeptide, fusion polypeptide, or carrier polypeptide may be linked to the C-terminus or N-terminus of the protease and may be directly attached to the protease, or may be separated from the protease by a linker peptide or polypeptide sequence.

[0026] Conservative variants of the NIa proteases, or its naturally occurring isoforms and homologs, are encompassed by the present invention. Such conservative mutations include mutations that switch one amino acid for another within one of the following groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

[0027] The types of substitutions selected may be based on the analysis of the frequencies of amino acid substitutions between homologous proteins of different species developed by Schulz et al. (Principles of Protein Structure, Springer- Verlag, 1978, pp. 14-16), on the analysis of structure-forming potentials developed by Chou and Fasman (Biochemistry 13, 211, 1974), or other such methods reviewed by Schulz et al. (Principles in Protein Structure, Springer-Verlag, 1978, pp. 108-130), and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doolittle (J. Mol. Biol. 157: 105-132, 1982).

[0028] The invention encompasses allelic variants having a slightly different amino acid sequence than the corresponding naturally occurring wild-type NIa protease or SEQ ID NO: 1 and having the requisite ability to recognize and cleave the heptapeptide sequence E-X-X-Y-X- QJ,S/G (SEQ ID NO: 20). The present invention also contemplates conservative variants that do not affect the ability of the protease to recognize and cleave the heptapeptide sequence E-X-X-Y- X-QJ,S/G (SEQ ID NO: 20). The present invention includes NIa proteases with altered overall charge, structure, hydrophobic/hydrophilic properties by amino acid substitutions, insertions, or deletions, but that still possess the ability to cleave the heptapetide recognition sequence. [0029] The present invention provides nucleic acid molecules comprising a sequence encoding a mutant protease of the invention. Polynucleotides encoding the proteases designated TEV1-TEV6 are described herein at SEQ ID NOs: 34-39. The polynucleotide encoding the wild-type TEV protease is described at Accession No. NC 001555 (SEQ ID NO: 1 is at NC_001555:6255..6980; SEQ ID NO: 2 is at NC_01555:5691..6980). The present invention also includes vectors, expression vectors, and host cells comprising a nucleic acid molecule encoding a mutated NIa protease.

[0030] Suitably, the nucleic acids encode mutated NIa proteases having the same as, similar or improved substrate cleavage activity to the corresponding wild-type NIa protease, or the 27 kDa wild-type TEV protease. The mutated NIa protease may have, for example, at least about 105%, 110%, 115%, 120%, 125%, 150%, 200% of the substrate cleavage activity as the corresponding wild-type NIa protease or the 27 kDa wild-type TEV protease. The nucleic acids may also encode mutated NIa proteases having, or having at least about, 50%>, 60%>,70%>, 75%>, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the corresponding wild-type NIa protease or of the 27 kDa wild-type TEV protease. Suitably, the nucleic acids will encode mutated NIa proteases having at least about 70%> sequence identity, at least about 80%> sequence identity, at least about 85%> sequence identity, at least about 90%> sequence identity, at least about 95%>, at least about 97%>, at least about 98%>, or at least about 99%> sequence identity to a wild-type NIa protease, such as TEV protease, or other naturally occurring isoforms having the same or similar substrate cleavage activity, or to one or more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,. SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0031] The term "homology" refers to a degree of complementarity between two or more sequences. There may be partial homology or complete homology (i.e., identity). The terms "homology" and "identity" in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. Methods of alignment of sequence for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith et al., (J. Mol. Evol. 18:38-46 (1981)), by the homology alignment algorithm of Needleman and Wunsch, (J. Mol. Biol, 48:443-453 (1970)), by the search for similarity method of Pearson and Lipman, (Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0032] Homology is often measured using sequence analysis software (e.g., "GCG" and "Seqweb" Sequence Analysis Software Package formerly sold by the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0033] The present invention also provides compositions comprising the mutated protease. The composition may include one or more of a buffer such as Tris, HEPES or phosphate butter; a detergent such as Triton present in an amount of about 0.01 to 1%, for example at least 0.05, 0.075, 0.09% or 0.1% and less than about 1%, 0.75%, 0.5%, 0.25% 0.2%, 0.15%; a reducing agent such as dithiothreital (DTT) present in an about of about 0.1 mM to 25 mM, suitably at least 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM or 0.75 mM and less than about 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 2 mM or 1 mM; a chelating agent such as ethylenediaminetetraacetic acid (EDTA) present in about 0.1 mM to 5 mM, suitably at least about 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM and less than about 5 mM, 4 mM, 3 mM, 2 mM, 1 mM; glycerol for example 25-75%) glycerol, suitably at least about 25%, 30%>, 35%, 40%>, or 45%), and less than about 75%, 70%, 65%, 60% or 55%; and a salt, such as sodium chloride (suitably in an amount of less than about 500 mM, 400 mM, 300 mM, 250 mM, 200 mM, 150 mM, 125 mM, 100 mM, 50 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, or 1 mM).

[0034] The present invention also provides methods of purifying or isolating a protein or polypeptide of interest using a mutated NIa protease described herein. Suitably, the protein or polypeptide of interest is linked to a polypeptide tag sequence, such as a His-tag, a HQ tag, GST tag, or a tag having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 19 (HaloTag^®), to form a tagged protein or polypeptide. The polypeptide tag sequence suitably has affinity for a resin or matrix. The tagged protein or polypeptide contains a cleavage site between the polypeptide tag and the polypeptide or protein of interest which is recognized by a NIa protease. SEQ ID NO: 20 is an example of such a cleavage site. The tagged protein or polypeptide is contacted with the resin or matrix such that it binds to the resin or matrix. Optionally the protein or polypeptide of interest bound to the resin or matrix is washed one, two, three or more times. The resin or matrix containing the bound tagged protein or polypeptide is contacted with the mutated NIa protease which cleaves the protein or polypeptide of interest from the tag, thereby releasing it from the resin or matrix.

[0035] The present invention further provides kits containing a mutated NIa protease. Suitably, the kits contain one or more mutated NIa proteases and one or more resins or matrices for binding a tagged polypeptide or protein or interest. The kits may also include the compositions or components of the compositions, described herein.

[0036] It will be apparent to those of skill in the art that variations may be applied to the compositions and methods described herein and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

[0037] It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0038] It also is understood that any numerical range recited herein includes all values from the lower value to the upper value. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10%> to 30%>, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. [0039] The following non-limiting examples are purely illustrative.

EXAMPLES

Example 1

[0040] Polynucleotide sequences encoding TEV1-TEV6 (SEQ ID NO: 4-9) were partially chemically synthesized by Beckman Coulter Genomics, with the C-terminus of the originally synthesized gene modified via PCR with oligonucleotides obtained from IDT (Iowa City, Iowa) to create the deletions.

[0041] Polynucleotide sequences TEV27vl-TEV27v6 were created by amplifying via PCR a template (SEQ ID NO: 36) with the following primers:

5^*Primer for all V1-V6: 5'GCGTGCGATCGCCTCGCTGTTTAAAGGCCCGCGT'3 (SEQ ID NO: 27)

3^* Primer for VI : 5 ATTAGTTTAAACTTACTGGCTATACACCAGTTCGTTCATCA3 (SEQ ID NO: 28)

3' Primer for V2:

5^*GATTGTTTAAACTTATTCTTCCGGTTTGCTCATAAACACTTTATGGC^*3 (SEQ ID NO: 29)

3^* Primer for V3: 5 TGTGTTTAAACTTACGGTTTGCTCATAAACACTTTATGGCCGC3 (SEQ ID NO: 30)

3^* Primer for V4: 5^*GTCGGTTTAAACTTATTTGCTCATAAACACTTTATGGCCGCCC^*3 (SEQ ID NO: 31)

3^* Primer for V5 5^*GTCGGTTTAAACTTAGCTCATAAACACTTTATGGCCGCCC^*3 (SEQ ID NO: 32)

3^* Primer for V6 5^*GTCGGTTTAAACTTACATAAACACTTTATGGCCGCCCC^*3 (SEQ ID NO: 33)

[0042] After amplification, the resulting products were cloned into the Flexi® Vector pFN18A (with deleted TEV protease site) using the Flexi® Cloning System. All clones were sequence verified by Sanger sequencing combined with a complete portfolio of Next Generation DNA sequencing technologies.

Example 2

[0043] Mutated NIa protease 500 was made using as a basis the sequence encoding the ProTEV protease (SEQ ID NO: 40; GST-linked ProTEV) by first removing the 5 C-terminal amino acids via synthetic oligonucleotides that span the BseYI-Xbal sites of TEV. Secondly, three point mutations, T17S, N68D, and I77V, were added by inserting a 224bp PCR product between the Nrul and Dral sites of TEV using mutagenic PCR primers. Mutated NIa proteases designated 501, 502 and 503 were made from the mutated NIa protease 500 by dropping in synthetic oligonucleotides between the Bell and EcoRI sites of TEV, using SEQ ID NO: 40 as a template.

[0044] The four mutants were expressed in Terrific Broth by IPTG induction at 25°C and allowed to grow overnight. The four mutant TEV proteins were purified on HisLink™ resin (commercially available from Promega) according to the manufacturer's protocol. Purified eluates were run on a 4-12% NuPAGE™ gel (commercially available from Invitrogen) and stained with SimplyBlue™ Safe Stain (commercially available from Invitrogen).

[0045] The results are shown in Figure 1. Mutant 500 (SEQ ID NO: 22) shows cleavage at the Vpg cleavage site resulting in a Vpg band at about 23 kDa. Mutant 501 (SEQ ID NO: 23) shows greatly reduced cleavage, and mutants 502 (SEQ ID NO: 24) and 503 (SEQ ID NO: 25) show no detectable cleavage.

Example 3

[0046] Mutated NIa proteases (SEQ ID NO: 4-15) were expressed in E. coli as HaloTag^® (HTv7) fusion proteins. Cells were lysed using sonication and cleared by centrifugation. The lysates were combined with SP6 High Yield Wheat Germ lysate (commercially available from Promega) expressing a circularly permuted firefly luciferase containing a TEV protease recognition sequence (Protease Glo Assay; Promega). Cleavage of the TEV protease recognition sequence leads to activation of the firefly luciferase and light emission. Activation of luciferase was detected using Bright-Glo™ Luciferase Assay Reagent (Promega) and measured using a luminometer. The lysates were run on a SDS-PAGE gel and the concentrations normalized. The normalized relative light units (RLU) were graphed to show mutated protease activity (Figures 2A and 2B).

Example 4

[0047] Mutated NIa protease (SEQ ID NO: 17) was expressed in E. coli. After cell lysis, the lysate was run on a Butyl HIC column. The fractions containing mutated NIa protease were combined and loaded on a Blue Sepharose column. The fractions containing mutated NIa protease were combined and loaded on an S-ion exchange column. The fractions containing mutated NIa protease were combined and then split into 3 pools. Each pool was dialyzed into either Buffer 4, 8, or 10 (Figure 3). After dialysis, aliquots were taken from each pool on days 1, 2 , 4, 6, and 7 and placed at 4°C, 22°C, 30°C, 37°C, and 44°C. The samples were then separated on a 4-20% SDS-PAGE gel (Figure 4), and the stability of each pool at each time point and temperature determined. In Figure 4, "lane C" depicts the control for each buffer, in which the HQ:TEV5 protein in the relevant buffer (4, 8, or 10), stored at -20°C, was loaded for comparison of cleavage with the same protein/buffer combination held at an elevated temperature. HQ:TEV5 shows superior stability to the protein lacking the deletion (ProTEV), regardless of buffer conditions.

Example 5

[0048] To demonstrate the improved solubility of the TEV mutants of the present invention, TEV3 and TEV5 HaloTag® fusion proteins were expressed in KRX cells (commercially available from Promega) using different induction conditions: 3 hour induction with Rhamnose at an OD₆₀o of 1.2; 24 hour induction with Rhamnose at an OD₆₀o of 1.2; early walk away induction (Schagat et al. Jan 2008. Promega Notes 98: 16-18) with 0.05% glucose and 0.05% Rhamnose; and late walk away induction (Schagat et al) with 0.15% glucose and 0.2% Rhamnose. Total and soluble fractions of the expressed proteins were labeled with HaloTag® TMR ligand (Promega) and analyzed on an SDS-Page gel. The expressed proteins were visualized fluorescently (Figure 5, top panel) and by Coomassie stain (Figure 5, bottom panel). The data demonstrates that HaloTag®-TEV5 is more soluble the HaloTag®-TEV3.

Description of Sequences in Sequence Listing

[0049] SEQ ID NO: 1 (Wild-type TEV 27 kDa catalytic domain -27.5 kDa)

[0050] SEQ ID NO: 2 (Wild-type 49 kDa TEV protease -48.7 kDa)

[0051] SEQ ID NO: 3 (wild-type Vpg domain -21.2 kDa) [0052] SEQ ID NO 4 (TEV 1 polypeptide)

[0053] SEQ ID NO 5 (TEV 2 polypeptide)

[0054] SEQ ID NO 6 (TEV 3 polypeptide)

[0055] SEQ ID NO 7 (TEV 4 polypeptide)

[0056] SEQ ID NO 8 (TEV 5 polypeptide -46.4 kDa)

[0057] SEQ ID NO 9 (TEV 6)

[0058] SEQ ID NO 10 (TEV27vl)

[0059] SEQ ID NO 11 (TEV27v2; catalytic domain of TEV5 -25.1 kDa)

[0060] SEQ ID NO 12 (TEV27v3)

[0061] SEQ ID NO 13 (TEV27v4 -24.8 kDa)

[0062] SEQ ID NO 14 (TEV27v5)

[0063] SEQ ID NO 15 (TEV27v6)

[0064] SEQ ID NO 16 (HaloTag® TEV5, 81.6 kDa)

Location/Feature

1..297 "HaloTag®"

496..503 = "mut cleavage site"

502..724 "TEV5"

313..501 "VpG"

298..312 = "linker peptide"

518 "T17S"

569 "N68D"

502..503 "G1V, E2N"

578 "I77V"

[0065] SEQ ID NO 17 (HQ-tagged TEV5)

[0066] SEQ ID NO 18 (HaloTag® -TEV27v2 - 60.0 kDa)

Location/Feature

1..297 "HaloTag®"

313..533 = "TEV5 catalytic domain"

313..501 "VpG"

298..312 = "peptide linker "

327 "T17S"

378 "N68D"

387 "I77V"

[0067] SEQ ID NO 19 (HaloTag® polypeptide) [0068] SEQ ID NO: 20 TEV Nla protease substrate recognition/cleavage site)

[0069] SEQ ID NO: 21 (Vpg site)

[0070] SEQ ID NO: 22 (ProTev500 polypeptide Nla protease)

[0071] SEQ ID NO: 23 (501 polypeptide Nla protease)

[0072] SEQ ID NO: 24 (502 polypeptide Nla protease)

[0073] SEQ ID NO: 25 (503 polypeptide Nla protease)

[0074] SEQ ID NO: 26 (TEV4 catalytic domain -25.9 kDa)

[0075] SEQ ID NO: 27 (primer)

[0076] SEQ ID NO: 28 (primer)

[0077] SEQ ID NO: 29 (primer)

[0078] SEQ ID NO: 30 (primer)

[0079] SEQ ID NO: 31 (primer)

[0080] SEQ ID NO: 32 (primer)

[0081] SEQ ID NO: 33 (primer)

[0082] SEQ ID NO: 34 (polynucleotide encoding TEV1)

[0083] SEQ ID NO: 35 (polynucleotide encoding TEV2)

[0084] SEQ ID NO: 36 (polynucleotide encoding TEV3)

[0085] SEQ ID NO: 37 (polynucleotide encoding TEV4)

[0086] SEQ ID NO: 38 (polynucleotide encoding TEV5)

[0087] SEQ ID NO: 39 (polynucleotide encoding TEV6)

[0088] SEQ ID NO: 40 (polynucleotide encoding ProTEV)

Location/Feature

1..654 "GST Tag"

655..699 = "peptide linker with TEV cleavage site"

700..2013 "ProTEV"

Claims

CLAIMS What is claimed is:

1. An isolated active mutant tobacco etch virus NIa protease wherein the amino acid residues corresponding to amino acids from position 231 to position 242 of SEQ ID NO: 1 are deleted.

2. The isolated active mutant NIa protease of claim 1, wherein amino acid residues corresponding to position 230 to position 242 of SEQ ID NO: 1, position 229 to position 242 of SEQ ID NO:. 1 , position 228 to position 242 of SEQ ID NO: 1, position 227 to position 242 of SEQ ID NO: 1, position 226 to position 242 of SEQ ID NO: 1, position 225 to position 242 of SEQ ID NO: 1, position 223 to position 242 of SEQ ID NO: 1, position 222 to 242 of SEQ ID NO: 1, position 221 to 242 of SEQ ID NO: 1, position 220 to 242 of SEQ ID NO: 1 or position 219 to 242 of SEQ ID NO: 1 are deleted.

3. The isolated active mutant NIa protease of claim 1, wherein the amino acid residues corresponding to amino acids from position 224 to position 242 of SEQ ID NO: 1 are deleted.

4. The isolated active mutant NIa protease of any one of claims 1 to 3, wherein the protease has increased proteolytic activity.

5. The isolated active mutant NIa protease of any one of claims 1 to 4, wherein at least about 50 % of the protease activity remains after storage at 4°C for 1 week.

6. The isolated active mutant NIa protease of any one of claims 1 to 5, wherein the amino acid corresponding to position 1 of SEQ ID NO: 1 is replaced with another amino acid, optionally a valine or a serine, the amino acid corresponding to position 2 of SEQ ID NO: 1 is replaced with another amino acid, optionally an asparagine or a combination thereof.

7. The isolated active mutant NIa protease of any one of claims 1 to 6, which does not comprise a Vpg domain, or portion thereof.

8. The isolated active mutant NIa protease of any one of claims 1 to 6, comprising at least a portion of a Vpg domain.

9. The isolated active mutant NIa protease of claim 8, wherein the at least a portion of a Vpg domain comprises six amino acid residues corresponding to position 183 to 188 of SEQ ID NO: 2 in which the residue corresponding to position 188 is replaced with another residue, optionally leucine, the residue corresponding to position 183 is replaced with another residue, optionally threonine or leucine, or a combination thereof.

10. The isolated active mutant NIa protease of any one of claims 1 to 9, wherein the amino acid residue corresponding to position 17 of SEQ ID NO: 1 is replaced with another residue, optionally serine, position 68 of SEQ ID NO: 1 is replaced with another residue, optionally aspartic acid, position 77 of SEQ ID NO: 1 is replaced with another residue, optionally valine, or a combination thereof.

11. The isolated active mutant NIa protease of any one of claims 1 to 10, wherein the protease has at least 80% identity with SEQ ID NO: 8 or SEQ ID NO: 11.

12. A polypeptide comprising the protease of any one of claims 1 to 11 linked to a polypeptide tag, the polypeptide tag selected from a metal binding tag, a His-tag, a HQ tag, a tag having at least 80% identity with SEQ ID NO: 19, a GST tag, or any combination thereof.

13. The polypeptide of claim 12, wherein the polypeptide tag is linked at the N-terminus of the protease, and optionally the polypeptide tag is separated from the protease by a polypeptide linker sequence.

14. The polypeptide of claim 12 or 13, wherein the polypeptide comprises SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

15. A composition comprising the protease of any one of claims 1 to 14 and further comprising DTT, EDTA, glycerol, detergent or a combination thereof.

16. A polynucleotide comprising a sequence encoding the protease of any one of claims 1 to 15.

17. A kit comprising the protease of any one of claims 1 to 11, the polypeptide of any one of claims 12 to 14, or the composition of claim 15, and a resin having the capacity to bind a polypeptide tag, optionally a His-tag, HQ tag, GST tag, or SEQ ID NO: 19.

18. A method of purifying a polypeptide linked to a polypeptide tag sequence, optionally a metal binding tag, a His-tag, a HQ tag, a GST tag, or a tag having at least 80% identity with SEQ ID NO: 19, optionally the tag being linked at the N-terminus of the protease, and optionally the tag being separated from the protease by a peptide or polypeptide linker sequence, the method comprising binding the polypeptide tag sequence to a matrix or resin and cleaving the polypeptide from the polypeptide tag sequence using the protease of any one of claims 1 to 11 or the polypeptide of any one of claims 12 to 14.