WO2011038219A1 - Protéases ayant une stabilité, une solubilité et une activité améliorées - Google Patents

Protéases ayant une stabilité, une solubilité et une activité améliorées Download PDF

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WO2011038219A1
WO2011038219A1 PCT/US2010/050184 US2010050184W WO2011038219A1 WO 2011038219 A1 WO2011038219 A1 WO 2011038219A1 US 2010050184 W US2010050184 W US 2010050184W WO 2011038219 A1 WO2011038219 A1 WO 2011038219A1
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seq
protease
tag
polypeptide
nia
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PCT/US2010/050184
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Brad Hook
Rachel Friedman Ohana
James Robert Hartnett
Michael R. Slater
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Promega Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • 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
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/34011—Potyviridae
    • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • 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.
  • TSV Tobacco Etch Virus
  • the protease cleaves its substrate between the Gin and Gly/Ser residues.
  • 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.
  • 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.
  • TSV tobacco etch virus
  • 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.
  • amino acids corresponding to position 224 to position 242 of SEQ ID NO: 1 are deleted.
  • 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.
  • 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).
  • 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.
  • 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.
  • TSV tobacco etch virus
  • 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-GloTM Assay (commercially available from Promega).
  • FIG. 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-GloTM Assay (commercially available from Promega).
  • 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.
  • 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.
  • Figure 5 shows photographs of protein gels showing the solubility of HaloTag®- TEV5 protease and HaloTag®-TEV3 protease.
  • 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.
  • the mutated proteases comprise an amino acid sequence in which a C-terminal portion of the protease is deleted.
  • 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.
  • 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.
  • 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.
  • 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.
  • NIa proteases are proteases that cleave the following peptide sequence Glu-Xaa 1 -Xaa 2 -Tyr-Xaa 3 -GlnJ,Xaa 4 (SEQ ID NO: 20) with cleavage indicated by the arrow, between the Gin and the Xaa 4 , where Xaa 1 , Xaa 2 and Xaa 3 , which may be the same or different, represent an amino acid and Xaa 4 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.
  • 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.
  • recognition sites include EDLVEQjS, EDLVEQjG, EIIYTQjS, EIIYTQjG, ETIYLQjS, ETIYLQjG, EPVYFQjS, EPVYFQjG, ELVYSQjS, ELVYSQjG, ENLYFQjS, and ENLYFQjG.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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, Wegn 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
  • the mutated NIa protease has a C-terminal deletion in the catalytic domain.
  • 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.
  • the mutated NIa protease may further include one or more mutations in combination with the C-terminal deletion.
  • 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.
  • 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.
  • 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.
  • 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.
  • the isolated active mutant NIa protease comprises at least a portion of or the entire Vpg domain.
  • 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.
  • 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,.
  • 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.
  • the present invention further provides polypeptides comprising mutated NIa proteases that are fused to a heterologous polypeptide, fusion polypeptide, or carrier polypeptide.
  • the heterologous polypeptide, fusion polypeptide, or carrier polypeptide facilitates isolation of the protease.
  • 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.
  • 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.
  • 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.
  • the heterologous polypeptide includes a glutathione S-transferase (GST) tag (position 1 to 654 of SEQ ID NO: 40).
  • 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.
  • 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:
  • Aromatic residues Phe, Tyr and Trp.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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
  • 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.
  • 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
  • the present invention also provides methods of purifying or isolating a protein or polypeptide of interest using a mutated NIa protease described herein.
  • 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.
  • 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.
  • kits containing a mutated NIa protease 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.
  • 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.
  • TEV1-TEV6 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.
  • Polynucleotide sequences TEV27vl-TEV27v6 were created by amplifying via PCR a template (SEQ ID NO: 36) with the following primers:
  • 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.
  • 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 HisLinkTM resin (commercially available from Promega) according to the manufacturer's protocol. Purified eluates were run on a 4-12% NuPAGETM gel (commercially available from Invitrogen) and stained with SimplyBlueTM Safe Stain (commercially available from Invitrogen).
  • Mutant 500 shows cleavage at the Vpg cleavage site resulting in a Vpg band at about 23 kDa.
  • Mutant 501 shows greatly reduced cleavage, and mutants 502 (SEQ ID NO: 24) and 503 (SEQ ID NO: 25) show no detectable cleavage.
  • 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.
  • luciferase activity was detected using Bright-GloTM 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).
  • 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).
  • 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 OD 60 o of 1.2; 24 hour induction with Rhamnose at an OD 60 o 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.
  • SEQ ID NO: 1 Wild-type TEV 27 kDa catalytic domain -27.5 kDa
  • SEQ ID NO: 2 Wild-type 49 kDa TEV protease -48.7 kDa
  • SEQ ID NO: 3 wild-type Vpg domain -21.2 kDa
  • SEQ ID NO 4 TSV 1 polypeptide
  • SEQ ID NO 8 (TEV 5 polypeptide -46.4 kDa)
  • SEQ ID NO 11 (TEV27v2; catalytic domain of TEV5 -25.1 kDa)
  • SEQ ID NO 16 (HaloTag® TEV5, 81.6 kDa)
  • SEQ ID NO 18 (HaloTag® -TEV27v2 - 60.0 kDa)
  • SEQ ID NO 19 HaloTag® polypeptide
  • SEQ ID NO: 20 TEV Nla protease substrate recognition/cleavage site
  • SEQ ID NO: 22 (ProTev500 polypeptide Nla protease)
  • SEQ ID NO: 23 (501 polypeptide Nla protease)
  • SEQ ID NO: 24 (502 polypeptide Nla protease)
  • SEQ ID NO: 25 (503 polypeptide Nla protease)
  • SEQ ID NO: 26 (TEV4 catalytic domain -25.9 kDa)
  • SEQ ID NO: 34 polynucleotide encoding TEV1
  • SEQ ID NO: 35 polynucleotide encoding TEV2
  • SEQ ID NO: 36 polynucleotide encoding TEV3
  • SEQ ID NO: 37 polynucleotide encoding TEV4
  • SEQ ID NO: 38 polynucleotide encoding TEV5
  • SEQ ID NO: 39 polynucleotide encoding TEV6
  • SEQ ID NO: 40 polynucleotide encoding ProTEV

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Abstract

L'invention porte sur des protéases NIa actives isolées, telles que celles provenant du virus de la gravure du tabac, qui sont modifiées par troncature de la protéase de telle sorte que les acides aminés C-terminaux de la protéase sont délétés. Les protéases NIa mutantes actives isolées peuvent présenter une ou plusieurs des caractéristiques suivantes: une activité protéolytique améliorée, une stabilité améliorée, moins de précipitation durant le stockage, un maintien de l'activité amélioré après le stockage, une solubilité améliorée. Les protéases NIa actives isolées peuvent comprendre la délétion C-terminale en combinaison avec d'autres modifications telles que des mutations ponctuelles. L'invention porte également sur des compositions et des kits contenant les protéases NIa mutantes isolées, et des procédés d'utilisation des protéases NIa mutantes isolées.
PCT/US2010/050184 2009-09-24 2010-09-24 Protéases ayant une stabilité, une solubilité et une activité améliorées WO2011038219A1 (fr)

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US9487520B2 (en) 2010-11-02 2016-11-08 Promega Corporation Coelenterazine derivatives and methods of using same
US9790537B2 (en) 2014-01-29 2017-10-17 Promega Corporation Quinone-masked probes as labeling reagents for cell uptake measurements
US9927430B2 (en) 2014-01-29 2018-03-27 Promega Corporation Pro-substrates for live cell applications
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Cited By (12)

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Publication number Priority date Publication date Assignee Title
JP2013524789A (ja) * 2010-04-16 2013-06-20 ヤンセン バイオテツク，インコーポレーテツド 改変された植物のシステインプロテアーゼ及びその使用
US8669103B2 (en) 2010-11-02 2014-03-11 Promega Corporation Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use
US9139836B2 (en) 2010-11-02 2015-09-22 Promega Corporation Imidazo[1,2-a]pyrazine derivatives
US9487520B2 (en) 2010-11-02 2016-11-08 Promega Corporation Coelenterazine derivatives and methods of using same
US9840730B2 (en) 2010-11-02 2017-12-12 Promega Corporation Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use
US9938564B2 (en) 2010-11-02 2018-04-10 Promega Corporation Substituted imidazo[1,2-a]pyrazines for use in bioluminogenic methods
US9951373B2 (en) 2010-11-02 2018-04-24 Promega Corporation Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use
US10774364B2 (en) 2010-11-02 2020-09-15 Promega Corporation Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use
US11661623B2 (en) 2010-11-02 2023-05-30 Promega Corporation Oplophorus-derived luciferases, novel coelenterazine substrates, and methods of use
US9790537B2 (en) 2014-01-29 2017-10-17 Promega Corporation Quinone-masked probes as labeling reagents for cell uptake measurements
US9927430B2 (en) 2014-01-29 2018-03-27 Promega Corporation Pro-substrates for live cell applications
WO2022093741A1 (fr) * 2020-10-26 2022-05-05 Research Development Foundation Protéases mutantes et leurs utilisations

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