Cold-active tev protease, compositions and methods

Abstract

According to some embodiments, the present disclosure relates to compositions, methods and kits for cleaving target proteins using cold-active variants of TEV protease.

Description

[0001] Sequence list declaration This disclosure contains an electronically submitted sequence list in .xml format, named “NEB-473.xml”, created on September 5, 2023, with a file size of 46.0 KB. The entire contents of this sequence list are incorporated herein by reference. Background Technology

[0002] Like other enzymes, the catalytic activity of proteases tends to increase with increasing temperature and decrease with decreasing temperature. For example, many enzymes can increase their activity by 50% to 100% when the reaction temperature increases by 10°C. Significant changes in activity (e.g., 10% or 20%) may be observed when the reaction temperature changes by 1°C or 2°C. This correlation between activity and reaction temperature may be limited to a certain temperature range, such as 15°C to 45°C. Higher reaction temperatures may result in a peak and subsequent decline in catalytic activity, which may be a result of protein denaturation, while lower temperatures may slow down or even reduce catalytic activity to zero, which may be a result of the enzyme becoming too rigid to interact with the substrate, perform the reaction, and / or release the product.

[0003] Proteases, including peptidases, are ubiquitous in prokaryotes and eukaryotes. For example, at least 14 superfamilies of cysteine ​​proteases have been described, each containing numerous protein families. Endopeptidases are proteases (proteolytic peptidases) that cleave peptide bonds between amino acids within a protein. The Tobacco Etch Virus (TEV) protease is a highly specific cysteine ​​protease used to remove affinity purification tags, such as maltose-binding protein (MBP) or polyhistidine, from fusion proteins. For example, TEV proteases can be used to cleave affinity tags from fusions comprising an affinity tag and a target protein (e.g., an enzyme). TEV proteases may exhibit peak activity at ~30°C, but the target protein may lack stability at this temperature. At lower temperatures, the target protein is (more) stable, and the TEV protease may exhibit reduced or no activity. Summary of the Invention

[0004] Therefore, there is a need for endopeptidases with enhanced activity at low temperatures. This disclosure relates to systems, apparatus, compositions, enzymes, and / or methods for cleaving target proteins at low temperatures. According to some embodiments, cold-active proteases may be more effective at cold temperatures than at warmer temperatures, and / or more effective than other proteases at the same cold temperature. For example, the cold-active variant TEV protease may have an amino acid sequence that is at least 90% identical to SEQ ID NO:1, wherein X49 is any amino acid except arginine, X73 is any amino acid except glutamine, X76 is any amino acid except leucine, X78 is any amino acid except aspartic acid, X82 is any amino acid except methionine, X83 is any amino acid except isoleucine, X84 is any amino acid except isoleucine, X89 is any amino acid except lysine, X102 is any amino acid except glutamic acid, X150 is any amino acid except glutamine, X155 is any amino acid except leucine, X181 is any amino acid except serine, X184 is any amino acid except lysine, X193 is any amino acid except glutamine, X206 is any amino acid except alanine, or X223 is any amino acid except glutamic acid. In some embodiments, the cold-active variant TEV protease may have an amino acid sequence that is at least 90% identical to SEQ ID NO:1, wherein at least one of X49, X73, X76, X78, X82, X83, X84, X89, X102, X150, X155, X181, X184, X193, X206, and X223 constitutes a substitution relative to the corresponding position in SEQ ID NO:31. For example, the cold-active variant TEV protease may have an amino acid sequence that is at least 98% identical to SEQ ID NO:2 or 3, or at least 97% identical to SEQ ID NO:20 or 21, or at least 95% identical to SEQ ID NO:11 or 12. In some embodiments, (a) X49 may be any amino acid other than arginine, or (b) X76 may be any amino acid other than leucine, or (c) X49 may be any amino group other than arginine and X76 may be any amino acid other than leucine. For example, (a) X49 may be phenylalanine or valine, or (b) X76 may be proline, or (c) X49 may be phenylalanine or valine and X76 may be proline. The amino acid sequence of the cold-active variant TEV protease may include one or more substitutions at position 45, a substitution at position 56, and a substitution at position 219 (in each case, the corresponding position relative to SEQ ID NO:31).According to some embodiments, the cold-active variant TEV protease may have an amino acid sequence including one or more of the following: K45Q substitution, K45R substitution, L56V substitution, S135G substitution, and S219V substitution. In some embodiments, the cold-active variant TEV protease may have an amino acid sequence wherein the amino acid sequence is located at positions 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 14... One or more of the following locations are identical to SEQ ID NO:1: 1, 146, 147, 148, 151, 153, 158, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, and 237.

[0005] According to some embodiments, the cold-active variant TEV protease may include an amino acid sequence having the following characteristics: (a) being at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1; (b) being substituted relative to SEQ ID NO:31 at positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223; (c) optionally being substituted at any position or any combination of positions corresponding to positions 56, 135, and 219 of SEQ ID NO:31; and (d) optionally being substituted at positions corresponding to SEQ ID NO:1. Position of NO:31: 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 141, 146, 147, 148, 151, 153, 15 The positions of 8, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, and 237, or any combination thereof, are identical to the wild-type TEV protease.

[0006] The cold-active variant TEV protease may include an amino acid sequence that, in some embodiments, (a) has at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:1, (b) has at least one substitution relative to the wild-type TEV protease (SEQ ID NO:31) at positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 corresponding to positions SEQ ID NO:31, (c) optionally has substitutions at any of positions 56, 135, and 219 or any combination thereof corresponding to positions SEQ ID NO:31, and (d) optionally has substitutions at positions corresponding to SEQ ID NO:1. NO:31 E2K, S3P, N12D, T17S, N23Q, H28L, H28Y, T30A, T30I, K45F, K45W, R50G, L56V, Q58F, Q58Y, Q58I, K67E, N68D, Q74L, I77V, G79E, R80S, M87L, M87T, D90G, P93S, E106G, E107D, C110S, K11 9E, S120R, S122P, D127A, C130S, F132L, F132S, S135G, S135F, I138T, K141R, T146A, T146C, T1 46S, K147E, D148R, D148P, D148A, C151A, S153C, S153N, T158A, F162S, F162A, S168T, S170A, N1 71D, N171Q, T173A, T173G, N176I, N176T, N177K, N177R, N177S, N177M, N177Y, T180A, R203Q, N 205D, V209M, V209F, W211I, W211V, W211L, W211C, K215E, V216I, F217K, M218I, M218F, M218W, M 218L, M218T, S219D, S219E, S219V, S219P, S219N, K220R, P224S, F225L, Q226stop, Q226S, Q226P, P227A, V228S, K229E, K229stop, M235K, N236S and E237G are missing one or more of the above substitutions at their respective positions; and (e) optionally, K67P, M82I, I83V, Q150D, Q150H, S153L, L155A, L155M and G213P corresponding to SEQ ID NO:31 are missing one or more of the above substitutions at their respective positions.

[0007] According to some embodiments, the cold-active variant TEV protease may have an amino acid sequence including a K45Q substitution or a K45R substitution and optionally one or more additional substitutions (e.g., L56V substitution, S135G substitution, and S219V substitution), in each case the substitution is at a position corresponding to the wild-type sequence. The cold-active TEV protease may have at least 90%, at least 92%, at least 95%, at least 97%, or at least 98% identity with one or more of SEQ ID NO: 9, 10, 18, 19, 27, and 28. For example, the cold-active TEV protease may have an amino acid sequence that is at least 98% identical to SEQ ID NO: 9 or 10, at least 97% identical to SEQ ID NO: 27 or 28, or at least 95% identical to SEQ ID NO: 18 or 19.

[0008] The provided variant TEV proteases may be more efficient than control TEV proteases (e.g., with higher catalytic efficiency). For example, the k of the variant TEV proteases (e.g., SEQ ID NO:1-28) cat / K M Compared with control TEV proteases (e.g., SEQ ID NO: 29-30) k cat / K M The ratio can be in the range of 1.3-3.0.

[0009] This disclosure further relates to a method for cleaving one or more TEV protease substrates. One method may include, for example, (a) contacting (i) a TEV protease substrate comprising a TEV protease recognition sequence having the amino acid sequence of SEQ ID NO:34, a target protein attached to the TEV protease recognition sequence, and a removable fragment attached to the TEV protease recognition sequence, and (ii) a variant TEV protease (e.g., any variant TEV protein disclosed herein) to form a mixture of cleavage products comprising the target protein and the removable fragment, respectively; and optionally separating the target protein from the removable fragment, wherein the contact may be performed at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

[0010] In some embodiments, a method may include causing (a) a composition comprising or possibly comprising a protein including a TEV protease recognition sequence having the amino acid sequence of SEQ ID NO:34, and (b) contacting a variant TEV protease (e.g., any variant TEV protease disclosed herein) to produce a cleavage product mixture, wherein the contact is performed at a temperature below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C, and / or wherein the cleavage product mixture optionally (i) does not contain the complete protein including the TEV protease recognition sequence (e.g., does not contain the complete protein within ≤3 hours, ≤5 hours, ≤12 hours, or ≤18 hours after contact), and / or (ii) contains at least two fragments of the protein including the TEV protease recognition sequence (e.g., within ≤1 hour, ≤3 hours, ≤5 hours, or ≤12 hours after contact).

[0011] A method may include (a) contacting (i) a TEV protease substrate comprising a TEV protease recognition sequence having the amino acid sequence of SEQ ID NO:34, a support attached to the TEV protease recognition sequence, and a decoy molecule attached to the TEV protease recognition sequence, and (ii) contacting a composition comprising a target molecule capable of binding the decoy molecule to form one or more TEV protease substrate:target molecule complexes. A method may include (e.g., may further include) (b) contacting one or more TEV protease substrate:target molecule complexes with a variant TEV protease (e.g., any variant TEV protease disclosed herein) to cleave the TEV protease substrate at the recognition sequence. (a) contacting, (b) contacting, or (a) and (b) contacting may be performed at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

[0012] The provided methods can use any desired variant TEV proteases disclosed herein, including, for example, variant TEV proteases that are more efficient (e.g., have higher catalytic efficiency) than the control TEV proteases. For example, the k-value of a variant TEV protease (e.g., any of SEQ ID NO: 1-28) cat / K M Compared with control TEV protease (e.g., SEQ ID NO: 29 or 30) k cat / K M The ratio can be in the range of 1.3-3.0. The variant TEV protease used in the disclosed methods, compositions or kits can be in any desired form, including, for example, soluble, lyophilized or immobilized forms.

[0013] This disclosure further relates to kits for cleaving target proteins (e.g., any target protein having a TEV protease recognition sequence). According to some embodiments, the kit may include a variant TEV protease (e.g., any variant TEV protease disclosed herein) and a buffer (e.g., a reaction buffer or storage buffer), wherein the variant TEV protease is in a form selected from liquid, gel, aqueous, film, crystal, powder, cake, dry, lyophilized, freeze-dried, and immobilized forms. Attached Figure Description

[0014] Figure 1 Examples of correlations between TEV protease concentration and fluorescence, representing peptide substrate cleavage activity, are shown.

[0015] Figure 2 The SDS-PAGE fractions of the products from the cell-free protein synthesis system are shown. Arrows indicate the expected positions of the TEV protease variants in each lane.

[0016] Figure 3 Examples of activity assay results obtained with some TEV protease variants, including those with proline substitutions, are shown. Arrows mark variants that exhibit higher activity than controls (TEV proteases with L56V, S135G, and S219V substitutions). Some variants exhibited activity comparable to the control group (e.g., variants including K184P, Q193P, A206P, or E223P).

[0017] Figure 4 Examples of activity assay results obtained with some TEV protease variants, including those with substitutions near His46, are shown. Arrows indicate variants that exhibited higher activity than the control.

[0018] Figure 5 Examples of activity assay results obtained with several TEV protease variants, including those with single or double substitutions, are shown. Arrows indicate variants that exhibited higher activity than the control.

[0019] Figure 6 The time-dependent cleavage of CBD-TEVrs-bglA by some TEV protease variants at 30 °C is shown.

[0020] Figure 7 The time-dependent cleavage of CBD-TEVrs-bglA by some TEV protease variants at 20 °C is shown.

[0021] Figure 8 The results of cleavage of the example CBD-TEVrs-bglA with some TEV protease variants are shown after 18 hours at 4°C.

[0022] Figure 9 Examples of activity assays obtained with some TEV protease variants, including those with bisubstituted amino acids, are shown. Arrows mark variants with a hydrophobic amino acid at position 49.

[0023] Figure 10 The time process of cleavage results obtained at 30°C using some example TEV protease variants is shown.

[0024] Figure 11 The cleavage results of the example CBD-TEVrs-bglA obtained after 18 hours at 4°C with 4 µM to 0.03125 µM (2-fold sequential) control and R49F_L76P variant TEV protease are shown. Under the test conditions, complete cleavage was observed with 2 µM control TEV protease and 0.5 µM R49F_L76P variant TEV protease.

[0025] Figure 12 The following figures show the cleavage results of example CBD-TEVrs-bglA obtained after 18 hours at 4°C (lanes 3, 4, and 5) or 0°C (lanes 6, 7, and 8) for some example TEV protease variants. The percentage of substrate cleavage is shown below each lane.

[0026] Figures 13A-13D The study showed an instance correlation between selected concentrations of the variant TEV protease and the cleavage rate of CBD-TEVrs-bglA after 18 hours at 4°C. Figure 13A Results for the R49V variant TEV protease are shown. Figure 13B Results for the L76P variant TEV protease are shown. Figure 13C Results for the R49V_L76P variant TEV protease are shown. Figure 13D Results for the R49F_L76P variant TEV protease are shown.

[0027] Figures 14A-14D Use respectively Figures 13A-13D The data show an example kinetic analysis of the TEV protease variant relative to the control TEV protease. Figure 14A The kinetic analysis of the R49V variant is shown, where k cat / K M The ratio is 2.0. Figure 14B The kinetic analysis of the L76P variant is shown, where k cat / K M The ratio is 1.7. Figure 14C The kinetic analysis of the R49V_L76P variant is shown, where k cat / K M The ratio is 1.5. Figure 14DThe kinetic analysis of the R49F_L76P variant is shown, where k cat / K M The ratio is 2.6.

[0028] Figure 15 The results of cleavage of example MBP5-TEV-paramyosin-ΔSal are shown using 0.125 µg of commercially available TEV protease (“Std”; New England Biolabs, catalog number #P8112S) and the R49F_L76P variant (“Cold”; SEQ ID NO:11) TEV protease at 4 °C for specified times (T0, T1, T3 and T5 hours).

[0029] Brief description of the sequence Some embodiments of this disclosure relate to sequences of example polynucleotides and / or example polypeptides provided below.

[0030] SEQ ID NO:1 is an example TEV protease variant having at least one substitution at a position selected from the following positions corresponding to SEQ ID NO:31: positions 45, 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223. Each X is independently any amino acid except that at least one of the following is true: 45 is not K, 49 is not R, 73 is not Q, 76 is not L, 78 is not D, 82 is not M, 83 is not I, 84 is not I, 89 is not K, 102 is not E, 150 is not Q, 155 is not L, 181 is not S, 184 is not K, 193 is not Q, 206 is not A, and / or 223 is not E. Although not marked with an "X", 56 is optionally not L (e.g., it is V instead of L), 135 is not S (e.g., it is G instead of S), and / or 219 is not S (e.g., it is V instead of S).

[0031] SEQ ID NO:2 is an example TEV protease variant with substitutions at positions 49 (R49F), 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49F_L76P".

[0032] SEQ ID NO:3 is an example TEV protease variant with substitutions at positions 49 (R49V), 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49V_L76P".

[0033] SEQ ID NO:4 is an example TEV protease variant with substitutions at positions 49 (R49F), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49F".

[0034] SEQ ID NO:5 is an example TEV protease variant with substitutions at positions 49 (R49V), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49V".

[0035] SEQ ID NO:6 is an example TEV protease variant with substitutions at positions 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "L76P".

[0036] SEQ ID NO:7 is an example TEV protease variant with substitutions (relative to the corresponding positions in SEQ ID NO:31) at positions 56 (L56V), 89 (K89P), 135 (S135G), and 219 (S219V). Enzymes including this sequence may be labeled "K89P".

[0037] SEQ ID NO:8 is an example TEV protease variant with substitutions (relative to the corresponding positions in SEQ ID NO:31) at positions 56 (L56V), 135 (S135G), 181 (S181P), and 219 (S219V). Enzymes including this sequence may be labeled "S181P".

[0038] SEQ ID NO:9 is an example TEV protease variant with substitutions (relative to the corresponding positions in SEQ ID NO:31) at positions 45 (K45Q), 56 (L56V), 135 (S135G), and 219 (S219V). Enzymes including this sequence may be labeled "K45Q".

[0039] SEQ ID NO:10 is an example TEV protease variant with substitutions (relative to the corresponding positions in SEQ ID NO:31) at positions 45 (K45R), 56 (L56V), 135 (S135G), and 219 (S219V). Enzymes including this sequence may be labeled "K45R".

[0040] SEQ ID NO:11 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 49 (R49F), 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled “R49F_L76P”.

[0041] SEQ ID NO:12 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 49 (R49V), 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled “R49V_L76P”.

[0042] SEQ ID NO:13 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 49 (R49F), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49F".

[0043] SEQ ID NO:14 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 49 (R49V), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49V".

[0044] SEQ ID NO:15 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 56 (L56V), 76 (L76P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "L76P".

[0045] SEQ ID NO:16 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 56 (L56V), 89 (K89P), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "K89P".

[0046] SEQ ID NO:17 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 56 (L56V), 135 (S135G), 181 (S181P), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "S181P".

[0047] SEQ ID NO:18 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 45 (K45Q), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "K45Q".

[0048] SEQ ID NO:19 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 45 (K45R), 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "K45R".

[0049] SEQ ID NO:20 is an example TEV protease variant with substitutions at positions 49 (R49F) and 76 (L76P) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "R49F_L76P". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:21 is an example TEV protease variant with substitutions at positions 49 (R49V) and 76 (L76P) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "R49V_L76P". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:22 is an example TEV protease variant with a substitution at position 49 (R49F) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "R49F". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:23 is an example TEV protease variant with a substitution at position 49 (R49V) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "R49V". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:24 is an example TEV protease variant with a substitution at position 76 (L76P) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "L76P". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:25 is an example TEV protease variant with a substitution at position 89 (K89P) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "K89P". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:26 is an example TEV protease variant with a substitution at position 181 (S181P) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "S181P". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:27 is an example TEV protease variant with a substitution at position 45 (K45Q) corresponding to SEQ ID NO:31. Enzymes including this sequence can be labeled "K45Q". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:28 is an example TEV protease variant with a substitution at position 45 (K45R) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "K45R". [WT@56 (L56), 135 (S135), and 219 (S219).] SEQ ID NO:29 is an example TEV protease variant with substitutions at positions 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. In some embodiments, enzymes including this sequence are labeled "control".

[0050] SEQ ID NO:30 is an example TEV protease variant having an N-terminal His tag and substitutions at positions 56 (L56V), 135 (S135G), and 219 (S219V) corresponding to SEQ ID NO:31. Enzymes including this sequence may be labeled "control".

[0051] SEQ ID NO:31 is an example of wild-type TEV protease. One or more of positions 45, 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, 223 may be substituted as disclosed herein.

[0052] SEQ ID NO:32 is an instance fluorescent peptide (TEV protease substrate) having instance recognition sequences at positions 1-6, wherein cleavage occurs between positions 5 and 6.

[0053] SEQ ID NO:33 is an example TEV protease substrate, wherein position 1 (E1) further includes a chitin-binding domain, and position 7 (G7) is linked to 6-phosphate-β-glucosidase A. In some embodiments, the substrate is labeled “CBD-TEVrs-bglA”.

[0054] SEQ ID NO:34 is the example TEV protease recognition sequence, wherein a cleavage occurs between positions Q5 and X6. X6 is glycine, or optionally X6 can be any of glycine, serine, alanine, methionine, cysteine, asparagine, tyrosine, lysine, aspartic acid, glutamine, phenylalanine, threonine, tryptophan, arginine, leucine, glutamic acid, isoleucine, or valine.

[0055] SEQ ID NO:35 is an example TEV protease substrate (“MBP5-TEV-paramyosin ΔSal”), which includes a maltose-binding protein domain and a paramyosin fragment linked by a TEV protease recognition sequence. Detailed Implementation

[0056] Modern biotechnology offers practitioners a choice of protein production methods, as well as the proteins that can be produced. Target proteins can be produced through cell-based workflows (e.g., bacterial, yeast, mammalian, baculovirus / insect cell systems) or cell-free workflows (e.g., NEB Corporation's PURExpress® and NEBExpress® protein synthesis systems, Ipswich, MA). In both cases, it may be desirable to produce proteins in a form that allows for isolation or purification from other molecules and substances. For example, proteins can be produced as fusion proteins comprising a purification tag (e.g., an affinity tag) and the target protein. After purification and / or any other desired processing, the target protein can be cleaved from the tag (or other fusion partner) using an endopeptidase. This disclosure relates to variant endopeptidases in some embodiments. Variant proteases can cleave polypeptides (e.g., fusion proteins) with greater efficiency and / or at lower temperatures than one or more reference proteases.

[0057] Overall considerations Various aspects of this disclosure can be understood from the provided descriptions, drawings, sequences, embodiments, section headings, and examples, none of which should be construed as limiting the full scope of this disclosure in any way. Therefore, the innovative content described herein should be understood in conjunction with the full scope and spirit of this disclosure.

[0058] Each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with components and / or features of any of the other embodiments without departing from the scope or spirit of this teaching. Throughout the disclosure, the length of the list of example species within a particular genus may vary in different places. A shortened list of species for convenience should not be construed as excluding example species listed elsewhere in the specification. Any described method may be performed in the order of the events described or in any other logically possible order. Unless expressly required otherwise herein, each component, feature, and method step disclosed herein is optional, and this disclosure contemplates embodiments in which each optional element may be expressly excluded. Therefore, this statement is intended as a prior basis for the use of exclusive terms such as "solely" and "only" when stating claim elements or using the "negative" limitation. It is also intended as a prior basis for the use of selective terms such as "optionally" in relation to the statement of one or more claim elements.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Nevertheless, for clarity and ease of reference, certain terms are defined herein for embodiments of this disclosure.

[0060] Sources of common terms and symbols may include: standard papers and texts such as Kornberg and Baker, DNA Replication, 2nd ed. (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Press, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Eckstein (ed.), Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait (ed.), Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., John Wiley and Sons, New York (1994); and Hale & Markham, the Harper Collins Dictionary of Biology, Harper Perennial, New York (1991), etc.

[0061] As used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “a” and “an” include plural references. For example, the term “a protein” refers to one or more proteins, i.e., one protein and multiple proteins.

[0062] A range of numbers includes numbers within a defined range. All numbers should be understood as including the midpoint of integers above and below them; that is, the number 2 includes 1.5-2.5, the number 2.5 includes 2.45-2.55, and so on. When sample numerical values ​​are provided, unless otherwise specified, each individual numerical value can represent the middle value of a range, and together they can represent the extreme values ​​of a range.

[0063] In the context of this disclosure, "buffer" and "buffering agent" refer to a chemical entity or composition that is itself resistant to changes in pH and, when present in solution, allows the solution to resist changes in pH when it comes into contact with a chemical entity or composition (e.g., an acid or a base) having a higher or lower pH. Examples of suitable non-natural buffers that can be used in the disclosed compositions, kits, and methods include HEPES, MES, MOPS, TAPS, tris(hydroxymethyl)methylglycine, and Tris. Other examples of suitable buffers that can be used in the disclosed compositions, kits, and methods include ACES, ADA, BES, N,N-di(hydroxyethyl)glycine, CAPS, carbonate / bicarbonate, CHES, citric acid, DIPSO, EPPS, histidine, MOPSO, phosphate, PIPES, POPSO, TAPS, and triethanolamine.

[0064] In the context of this disclosure, "catalytically active" refers to the property of a molecule (e.g., a protein molecule or macromolecule) to act as a catalyst for one or more chemical reactions with respect to one or more substrates and products. Catalytically active TEV proteases, for example, hydrolyze substrate peptides to produce (even if only transiently, e.g., in the context of a coupling reaction) a product comprising at least two fragments of the substrate peptide. The catalytic activity of TEV proteases can be evaluated using existing techniques applied to one or more model substrates (e.g., SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:35) and / or one or more target substrates. For example, an efficient determination of the catalytic activity of a TEV protease may include: (1) cleaving an indicator peptide comprising a fluorophore, a quencher operatively linked to the fluorophore to quench the fluorophore, and a TEV protease recognition site disposed between the fluorophore and the quencher, wherein cleavage is assessed by the appearance and / or intensity of fluorescence released by the cleaved peptide; and (2) cleaving a fusion protein comprising a first protein, a second protein, and a TEV protease recognition site disposed between the first and second proteins, wherein cleavage is assessed by the appearance and / or intensity of bands corresponding to the first and / or second proteins after reaction product size chromatography (e.g., SDS-PAGE). Catalytic activity may be assessed based on the loss of the original substrate peptide (e.g., the percentage of the original peptide remaining), the appearance of one or more cleavage products (e.g., fragments of the substrate peptide), and / or as an indicator of any of the above substitutes.

[0065] In the context of this disclosure, "catalytic efficiency" refers to the rate of reaction catalyzed by a catalyst (e.g., a variant TEV protease), denoted as k.cat or k cat / K M 。

[0066] In the context of the present disclosure, a "container" refers to an artificial container. A container can include one or more walls (e.g., defining an internal volume) and optionally one or more openings. A container including one or more openings can also include one or more closures (e.g., detachable closures) for some or all of these openings. The closure can optionally include a hole or diaphragm, e.g., to provide fluid communication with a volume of the container and an inserted tube or syringe. Examples of containers include boxes, cartons, bottles, tubes (such as test tubes, microcentrifuge tubes), plates (such as 96-well, 384-well plates), vials, pipette tips, and ampoules. The container and / or closure can include any desired material, including paper, plastic, glass, silicone, composite materials, metals, alloys, or combinations thereof. The container and / or closure can include compostable, recyclable, and / or sustainable materials.

[0067] In the context of the present disclosure, a "cold-active" enzyme refers to an enzyme that has catalytic activity at cold temperatures (e.g., at a temperature within the range of X to Y, where X is any one of -4°C, -2°C, -2°C, 0°C, 2°C, 4°C, and 8°C, and Y is any one of 0°C, 2°C, 4°C, 8°C, 10°C, 15°C, 18°C, and 20°C and X < Y). A cold-active enzyme may or may not have catalytic activity at warmer temperatures. For example, under the same reaction conditions as the cold conditions, a cold-active enzyme can have catalytic activity (e.g., >0%, ≤25%, ≤50%, ≤75%, ≤100%, ≥110%, ≥125%, ≥150%, ≥175%, or ≥200% of its cold activity) at a warmer temperature (e.g., at a temperature within the range of X' to Y', where X' is any one of 10°C, 15°C, 18°C, and 20°C, and Y' is any one of 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C and X' < Y'). A cold-active enzyme can react with the same, similar, or different kinetics at colder and warmer temperatures. A cold-active enzyme can react through the same, similar, or different reaction mechanisms at colder temperatures compared to warmer temperatures. If an enzyme can exhibit trace activity at colder temperatures (e.g., 0°C - 4°C), and this trace activity depends on an excess of the reaction with the enzyme (e.g., ≥10-fold, ≥20-fold, ≥30-fold, ≥40-fold, ≥50-fold of that required at higher temperatures) and / or depends on the use of an overly long incubation time (e.g., ≥5-fold, ≥10-fold of that required at higher temperatures), then the sole possibility that the enzyme can exhibit trace activity at colder temperatures (e.g., 0°C - 4°C) is not sufficient to be considered a cold-active enzyme.

[0068] In the context of this disclosure, for the position of an amino acid residue or nucleotide base, "corresponding to" means a position relative to another when sequence alignment is performed, for example by the BLAST algorithm. An amino acid position in a functional or structural motif of one polymerase may correspond to a position in a functionally equivalent functional or structural motif of another polymerase.

[0069] In the context of this disclosure, "fusion" refers to the covalently linked (e.g., via peptide bonds) two or more polypeptides, subunits, or proteins. For example, protein fusion can refer to a non-naturally occurring polypeptide comprising a target protein covalently linked to a second polypeptide. Examples of the second polypeptide include reporter proteins, purification and expression tags, polynucleotide-binding proteins, enzymes, conjugation tags (e.g., SNAP® tags), and peptide linkers. Unless otherwise disclosed, the target protein may be closer to the N-terminus or C-terminus than the second polypeptide to which it is linked. Fusions can include non-naturally occurring single polypeptide chains comprising two proteins or two protein domains linked directly to each other via peptide bonds or via peptide linkers (e.g., cleavable or non-cleavable linkers). Fusions may include variant TEV proteases.

[0070] In the context of this disclosure, "immobilized" refers to an enzyme covalently attached to a solid support with or without a linker. Examples of solid supports include beads (e.g., magnetic beads, agarose, polystyrene, polyacrylamide, chitin). Beads may include one or more surface modifications (e.g., O...). 6 (e.g., benzylguanine, polyethylene glycol) to promote covalent attachment and / or activity of the target enzyme. For example, the support may include a ligand, and the enzyme may have a receptor for such a ligand, or the enzyme may include a ligand and the support may include a receptor for such a ligand. Receptor-ligand binding can be covalent or non-covalent. Non-covalent attachment (e.g., avidin:biotin, chitin:CBP) may be useful in some embodiments, for example, where the dissociation level of the binding chaperone protein is considered acceptable. A linker may be positioned between the support and the enzyme. For example, a linker positioned between the support and the enzyme may have a first covalent bond with the support and a second covalent bond with the enzyme. Immobilized enzymes including ligand-receptor attachments may have a linker positioned between the support and the ligand-receptor attachment, a linker positioned between the enzyme and the ligand-receptor attachment, or both. Immobilized enzymes including linkers may also include an optional covalent bond directly between the enzyme and the support. The linker may have any desired length and any desired range of motion. Peptide linkers may include one or more repeats of glycine-serine (e.g., 1-10 repeats).

[0071] In the context of this disclosure, "non-naturally occurring" means a molecule (e.g., a polynucleotide, polypeptide, carbohydrate, or lipid) or composition that does not exist in nature. Such a molecule or composition may differ from naturally occurring molecules or compositions in one or more respects. For example, the types and arrangements of the components (e.g., nucleotide sequences, amino acid sequences, or sugar molecules) of a polymer (e.g., a polynucleotide, polypeptide, or carbohydrate) may differ. The polymer may differ from naturally occurring polymers in terms of the molecules it links. For example, a "non-naturally occurring" polypeptide (such as a protein) may differ from a naturally occurring polypeptide in its secondary, tertiary, or quaternary structure because it has (or lacks) chemical bonds (e.g., covalent bonds, including peptide bonds, phosphate bonds, disulfide bonds, ester bonds, and ether bonds, etc.) with lipids, carbohydrates, secondary polypeptides (such as fusion proteins), or any other molecule. Similarly, a "non-naturally occurring" polynucleotide or nucleic acid may include (or lack) one or more other modifications (e.g., added tags or other moieties) at the 5' end, 3' end, and / or between the 5' and 3' ends of the nucleic acid (e.g., methylation). A “non-naturally occurring” molecule or composition may differ from a naturally occurring composition in one or more of the following ways: (a) it has a component that is not bound in nature; (b) it has a component in proportions and / or concentrations not found in nature; (c) it lacks one or more components that are normally present in naturally occurring molecules or compositions (e.g., cell-free compositions, chromosome-free compositions, histone-free compositions, polymerase-free compositions, cell membrane-free compositions); (d) it has a form not found in nature (e.g., dried, freeze-dried, lyophilized, crystallized, aqueous, immobilized); and (e) it has one or more other components not found in nature (e.g., buffers, surfactants, dyes, solvents, or preservatives).

[0072] In the context of this disclosure, an amino acid sequence having a percentage identity with a reference sequence may be disclosed, wherein one or more variant positions may be specified or not specified. For example, if a 100-amino acid polypeptide is disclosed as having an amino acid sequence having at least 90% identity with a 100-amino acid reference sequence, then the sequence may differ from the reference sequence at any of up to 10 positions. If a 100-amino acid polypeptide is disclosed as having an amino acid sequence having at least 90% identity with a 100-amino acid reference sequence and having a K49R substitution, then the sequence may differ from the reference sequence at position 49 and at any of up to 9 other positions as shown.

[0073] In the context of this disclosure, with respect to an amino acid, "position" refers to the numbered position of that amino acid in the primary sequence of a peptide or polypeptide, from the amino terminus to the carboxyl terminus.

[0074] In the context of this disclosure, "substitution" means that an amino acid residue at a position in a comparative amino acid sequence differs from the corresponding position in a reference amino acid sequence, wherein the comparative and reference sequences are at least 60% identical to each other, or at least 70% identical to each other, or at least 80% identical to each other. The lengths of the reference and comparative sequences may be the same or similar (e.g., differing by ≤12%, ≤5%, ≤1%). In addition to differing from the corresponding position in the reference amino acid sequence, the substituted amino acid residue at the position may also differ from the corresponding position in all naturally occurring sequences that are at least 60% identical to each other, or at least 70% identical to each other, or at least 80% identical to each other. Optionally, the substituted amino acid may have properties different from the amino acid at the corresponding position in the reference sequence. Optionally, the substituted amino acid may have properties similar to the amino acid at the corresponding position in the reference sequence ("conservative" substitution). For example, nonpolar amino acids (e.g., A, V, L, I, M, W, and F (and optionally C, G, and P)) can replace another nonpolar amino acid; polar amino acids (e.g., N, Q, S, T, and Y) can replace another polar amino group (e.g., C, D, E, H, K, N, P, Q, R, S, and T); positively charged amino acids (e.g., H, K, and R) can replace another positively charged amino acid; and negatively charged amino acids (e.g., D and E) can replace another negatively charged amino acid. The substituted amino acid can be a natural amino acid (e.g., replacing another natural or non-natural amino acid). The substituted amino acid can also be a non-natural amino acid (e.g., replacing one natural or another non-natural amino acid).

[0075] In the context of the present disclosure, "variant TEV protease" refers to a non-naturally occurring enzyme that hydrolyzes peptide bonds of one or more polypeptide substrates. The variant TEV protease can recognize, bind to, and / or cleave polypeptide substrates that include the amino acid sequence ENLYQX (SEQ ID NO:34), where X can be glycine, or optionally any one of serine, alanine, methionine, cysteine, asparagine, histidine, tyrosine, lysine, aspartic acid, glutamine, phenylalanine, threonine, tryptophan, arginine, leucine, glutamic acid, isoleucine, or valine, and where the cleaved peptide bond is between amino acids Q5 and X6. The variant TEV protease can have catalytic activity upon exposure to and / or following exposure to temperatures within the range of X to Y, where X is any one of -4°C, -2°C, -2°C, 0°C, 2°C, 4°C, 8°C, and Y is any one of 0°C, 2°C, 4°C, 8°C, 10°C, 15°C, 18°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and X < Y. The variant TEV protease can cleave polypeptide substrates more efficiently than a reference protease, such as wild-type TEV protease (SEQ ID NO:31), at any given temperature. For example, at 4°C, the increase in cleavage efficiency of the variant TEV protease compared to wild-type TEV protease can be within the range of X'' to Y'', where X'' is any one of 5%, 10%, 25%, 50%, 75%, or 100%, and Y'' is any one of 10%, 25%, 50%, 75%, 100%, 250%, 500%, or 1000% (e.g., 5%-25%, 5%-100%, 10%-250%, 25%-1000% and X'' < Y'').

[0076] The catalytic activity of the variant TEV protease can persist over a range of salt concentrations, temperatures, and / or pH values. For example, the variant TEV protease can exhibit catalytic activity under a range of conditions and / or following removal from these conditions. The variant TEV protease can have catalytic activity upon exposure to to the pH values of, where is any one of pH 4, 4.5, 5, 5.5, 6, 6.5, 7, and is any one of pH 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 and < .

[0077] Variant TEV proteases may have an amino acid sequence that shares any desired degree of sequence identity with wild-type TEV proteases (SEQ ID NO:31), up to (but not including) 100% identity (237 / 237). Variant TEV proteases may include amino acid sequences having at least one substitution (e.g., at least two, at least three, or at least four substitutions) relative to wild-type TEV proteases at positions 45, 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 corresponding to positions SEQ ID NO:31. Apart from such substitutions, variant TEV proteases may be identical to wild-type TEV proteases (e.g., SEQ ID NO:31). For example, variant TEV proteases may include amino acid sequences characterized by: (a) Has at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% identity with SEQ ID NO:1. (b) At least one substitution relative to wild-type TEV protease (SEQ ID NO:31) at positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 corresponding to SEQ ID NO:31. (c) Optionally, it may be replaced at any of the positions 56, 135, and 219 corresponding to SEQ ID NO:31, or any combination thereof, and (d) Optionally, at positions 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 141, 146, 147, 148, 151, 153, 1 corresponding to SEQ ID NO:31 At any position or any combination of positions 58, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, 237, the protease is identical to that of wild-type TEV at any position or combination of positions.

[0078] In some implementations, it may be desirable to avoid certain substitutions at certain positions. For example, variant TEV proteases may include the following amino acid sequence: (a) Has at least 85%, at least 90%, at least 95%, at least 97%, or at least 98% identity with SEQ ID NO:1. (b) At the position corresponding to SEQ ID NO:31 45 The positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 have at least one substitution relative to the wild-type TEV protease (SEQ ID NO: 31), wherein if the at least one substitution includes a substitution at position 45, the substitution at position 45 is selected from K45Q and K45R; (c) Optionally, it may be replaced at any of the positions 56, 135, and 219 corresponding to SEQ ID NO:31, or at any combination of those positions. (d) Optionally, E2K, S3P, N12D, T17S, N23Q, H28L, H28Y, T30A, T30I, K45F, K45W, R50G, L56V, Q58F, Q58Y, Q58I, K67E, N68D, Q74L, I77V, G79E, R80S, M87L, M87T, D90G, P93S, E106G, E107D, C110S, K119E corresponding to SEQ ID NO:31 , S120R, S122P, D127A, C130S, F132L, F132S, S135G, S135F, I138T, K141R, T146A, T146C, T146S, K147E, D148R, D148P, D148A, C151A, S153C, S153N, T158A, F162S, F162A, S168T, S170A, N171D, N 171Q, T173A, T173G, N176I, N176T, N177K, N177R, N177S, N177M, N177Y, T180A, R203Q, N205D, V2 09M, V209F, W211I, W211V, W211L, W211C, K215E, V216I, F217K, M218I, M218F, M218W, M218L, M21 The following conditions are missing one or more substitutions (e.g., two or more, three or more, or all of the above substitutions) at the positions of 8T, S219D, S219E, S219V, S219P, S219N, K220R, P224S, F225L, Q226stop, Q226S, Q226P, P227A, V228S, K229E, K229stop, M235K, N236S, and E237G; and (e) Optionally, one or more substitutions (e.g., two or more, three or more, or all of the above substitutions) are omitted at the positions corresponding to K67P, M82I, I83V, Q150D, Q150H, S153L, L155A, L155M and G213P of SEQ ID NO:31.

[0079] In some embodiments, the variant TEV protease may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:1, wherein at least one of X45, X49, X73, X76, X78, X82, X83, X84, X89, X102, X150, X155, X181, X184, X193, X206, and X223 relative to SEQ ID NO:1. The corresponding position of NO:31 constitutes a substitution, for example, where X45 is any amino acid other than lysine (e.g., K45Q, K45R), where X49 is any amino acid other than arginine, X73 is any amino acid other than glutamine, X76 is any amino acid other than leucine, X78 is any amino acid other than aspartic acid, X82 is any amino acid other than methionine, X83 is any amino acid other than isoleucine, X84 is any amino acid other than isoleucine, X89 is any amino acid other than lysine, X102 is any amino acid other than glutamic acid, X150 is any amino acid other than glutamine, X155 is any amino acid other than leucine, X181 is any amino acid other than serine, X184 is any amino acid other than lysine, X193 is any amino acid other than glutamine, X206 is any amino acid other than alanine, and / or X223 is any amino acid other than glutamic acid. The variant TEV protease may optionally include at least one additional substitution (relative to the corresponding position of SEQ ID NO:31). For example, the variant TEV protease may have amino acid sequences with one or more substitutions at positions 56, 135, and 219 relative to SEQ ID NO:31. The variant TEV protease may also optionally include one or more of the above substitutions, such as L56V, S135G, and S219V relative to SEQ ID NO:31.The variant TEV protease may optionally also be present at positions 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 141, 146, 147, 148, 151 corresponding to SEQ ID NO:31. One or more of the following positions are identical to SEQ ID NO:31: 153, 158, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, and 237.

[0080] According to some embodiments, the variant TEV protease may have at least one substitution relative to the wild-type TEV protease (SEQ ID NO:31) and / or may be identical to one or more amino acids of the wild-type TEV protease (SEQ ID NO:31). Table 1 shows examples of positions corresponding to the wild-type TEV protease (SEQ ID NO:31), which may be identical or have substituted amino acids (in each case relative to the wild-type). The indicated position can be a single amino acid substitution, the same amino acid with an N-terminal extension (e.g., a label, signal peptide, purification tag), or a single amino acid substitution that further includes an N-terminal extension (e.g., a label, signal peptide, purification tag).

[0081] The indicated position can be a single amino acid substitution, the same amino acid having a C-terminal extension (e.g., a tag, peptide, purification tag), or a single amino acid substitution that further includes a C-terminal extension (e.g., a tag, peptide, purification tag).

[0082] In some embodiments, the variant TEV protease may have an amino acid sequence according to any one of SEQ ID NOS:1-30. In some embodiments, the variant TEV protease may have an amino acid sequence according to any one of SEQ ID NOS:1-28. The variant TEV protease fusion may have an amino acid sequence comprising a first portion and a second portion, the first portion or the second portion comprising the variant TEV protease (e.g., a variant TEV protease having any one of SEQ ID NO:1-28).

[0083] According to some implementations, the variant TEV protease may exhibit higher catalytic efficiency than the control TEV protease. For a given substrate (e.g., CBD-TEVrs-bglA or any other protein including TEVpRS) and at a given temperature (e.g., 4°C), the kJ / kJ of the variant TEV protease... cat / K M Compared with the control TEV protease k cat / K M The ratio, for example, can be found in X a To Y b Within the range, where X a For any one of 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, or 2.4, and Y b It is any one of 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 and X a <Y b For example, the variant TEV protease versus the control TEV protease kJ cat / K M The ratio can be in the range of 1.3-3.0, 1.4-2.9, 1.5-2.8, or 1.6-2.7.

[0084] In some embodiments, one unit of the variant TEV protease can cleave 2 µg of the MBP fusion protein MBP5-TEV paramyosin ΔSal to 95% in 1 hour at 30°C in 50 mM Tris-HCl (pH 7.5 @ 25°C) with 0.5 mM EDTA and 1 mM DTT in a total reaction volume of 10 µl over 1 hour.

[0085] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference. The reagents referenced in this disclosure can be prepared using available materials and techniques obtained from indicated sources and / or from New England Biolabs (Ipswich, MA).

[0086] Enzymes and Compositions In some embodiments, this disclosure relates to a means of cleaving a target protein at cold temperatures (e.g., ≤20°C, ≤18°C, ≤16°C, ≤15°C, ≤14°C, ≤12°C, ≤10°C, ≤8°C, ≤6°C, ≤5°C, ≤4°C, ≤3°C, and / or ≤2°C). Examples of means of cleaving a target protein at cold temperatures include all variant TEV proteases having the physical and chemical properties (e.g., sequence, motif, bond, binding properties, and other structural and feature characteristics) disclosed herein. In some embodiments, this disclosure relates to variant TEV proteases having one or more desired properties, including, for example, efficient, selective, and cold-active cleavage of one or more target proteins relative to, for example, wild-type TEV proteases.

[0087] In some embodiments, this disclosure relates to an immobilized enzyme comprising a support and an enzyme immobilized thereon. For example, an immobilized variant TEV protease may include a variant TEV protease, a glycine-serine linker attached to the variant TEV protease via a peptide bond, a protein tag (e.g., a SNAP tag) attached to the linker via a peptide chain, and an O-type linker bound to the protein tag (e.g., a SNAP-tag®). 6 -benzylguanine; and having O 6 - Surface-modified beads (e.g., magnetic beads) of benzylguanine. In some embodiments, the support for the immobilized variant TEV protease may include magnetic beads. Magnetic beads may include, for example, one or more surface modifications. Surface modifications may include, for example, O 6 -Benzylguanine and / or PEG 750 In some embodiments, the immobilized enzyme may include a ligand (e.g., O). 6 (-Benzylguanine) and a receptor or tag (e.g., SNAP-tag®) capable of binding the ligand. For example, the ligand may be disposed on a support, and the corresponding receptor may be disposed (e.g., covalently attached to) the enzyme to be immobilized on the support. In some embodiments, the immobilized enzyme may include an enzyme (e.g., a variant TEV protease), optionally, attached to a first linker (e.g., a peptide linker), attached to the first linker (if present) or a polypeptide tag of said enzyme (e.g., SNAP-tag®), and attached (e.g., covalently attached to) a ligand (e.g., O) corresponding to the polypeptide tag to the tag. 6 -benzylguanine), optionally, a second connector (e.g., polyethylene glycol) attached to the ligand, and a support (e.g., a magnetic bead) attached to the second connector (if present) or the ligand, the structure of which can be shown in the N->C direction as follows: Enzyme – [Connector –] Tag – Ligand – [Connector –] Support or Support – [Connector –] Ligand – [Connector –] Enzyme (collectively referred to as "iA") The short lines represent bonds (covalent or non-covalent), and the brackets represent optional elements.

[0088] According to some embodiments, the variant TEV protease composition may include a variant TEV protease and optionally include any of the following: a buffer (e.g., storage buffer, reaction buffer), an excipient, a salt (e.g., NaCl, MgCl2, CaCl2), a protein (e.g., an internal control with or without a TEV protease recognition sequence), a stabilizer, a detergent (e.g., ionic, nonionic, and / or zwitterionic detergents (e.g., octanylbenzyl alcohol, polysorbate 20), a polynucleotide, cells (e.g., whole, digested, or any cell-free extract), a biological fluid or secretion (e.g., mucus, pus), an aptamer, a pH indicator (e.g., zolidinium, bromophenol violet, bromocresol blue, methylene blue, cresol red, neutral red, naphtholphthalein, phenol red), a clustering agent, a sugar (e.g., monosaccharide, disaccharide, diacetylene, chloroform ... Sugars, trisaccharides, tetrasaccharides, or higher sugars), starch, cellulose, glass-forming agents (such as glycerol, raffinose, stachyose, or trehalose for lyophilization), lipids, oils, aqueous media, supports (such as beads), and / or combinations thereof (not naturally occurring). Combinations may include, for example, two or more of the listed components (e.g., salts and buffers) or multiple species of a single listed component (e.g., two different salts or two different sugars). According to some embodiments, variant TEV protease compositions may include (a) a variant TEV protease and (b) a protein (e.g., a known variant TEV protease substrate, a candidate variant TEV protease substrate, a test sample including or possibly including a variant TEV protease substrate), or a cell extract or cell-free formulation including a variant TEV protease substrate.

[0089] The variant TEV protease composition may include, for example, a variant TEV protease (e.g., having an amino acid sequence that is at least 85% identical to one or more of the sequences in SEQ ID NO: 1-28) and having at least one substitution relative to the wild-type TEV protease. The variant TEV protease composition may not have one or more other catalytic activities. For example, the variant TEV protease may be free of other proteases (e.g., nonspecific proteases or proteases with other cleavage recognition sites), free of nucleases (e.g., RNase and / or DNase), free of polymerase activity, free of RNA and / or DNA modification activity, free of kinase activity, and / or free of phosphorylation and / or glycosylation activity, in each case, under desired test conditions (e.g., time, temperature, pH, salinity, model or expected substrate conditions and / or other conditions), for example, under conditions intended to replicate the specific use of the variant TEV protease composition or under conditions intended to represent a range of uses.

[0090] In some embodiments, the variant TEV protease and the composition comprising one or more variant TEV proteases can be in any desired form, including, for example, liquid, gel, film, powder, cake, and / or any dried or lyophilized form. The variant TEV protease composition may include the variant TEV protease and a support or matrix, such as a film, gel, fabric, column, or bead, comprising, for example, magnetic materials, agarose, polystyrene, polyacrylamide, and / or chitin. The variant TEV protease and the composition comprising the variant TEV protease may be active at higher temperatures. For example, the variant TEV protease or the variant TEV protease composition may exhibit variant TEV protease activity at temperatures below 25°C to 50°C. In some embodiments, the variant TEV protease may exhibit cryogenic activity and / or psychrophilia. For example, the variant TEV protease or the variant TEV protease composition may exhibit variant TEV protease activity at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C. The composition may be in a fluid state at temperatures close to or below 0°C, for example, when the melting temperature of the composition is below a desired temperature. Aqueous compositions may include, for example, one or more elements that lower the melting temperature of the composition, including, for example, DMSO, methanol, glycerol, ethylene glycol, propylene glycol, sugars, amino acids, and proteins, etc.

[0091] In some embodiments, the variant TEV protease may be encoded by a nucleic acid sequence that, upon transcription, translation, and / or processing, produces an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 91%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1-30 (e.g., 1-28) and includes one or more substituted amino acid sequences relative to SEQ ID NO:31. The nucleic acid encoding the variant TEV protease can be contained in an expression cassette, expression vector, or other expressible form suitable for in vitro or in vivo expression (e.g., in *E. coli* or other bacteria, or *Pichia pastoris* or other yeasts). The nucleic acid encoding the variant TEV protease can be modified or optimized (e.g., codon optimization) for expression in the desired organism or cell-free expression system.

[0092] Methods and Workflow The variant TEV proteases disclosed herein can be used in a variety of methods and / or workflows, including manufacturing, molecular, cellular, research, sequencing, screening, diagnostic, and / or therapeutic applications. For example, the variant TEV proteases can be used in any application requiring the cleavage of molecules (e.g., proteins) that include a TEV protease recognition sequence (e.g., SEQ ID NO:34). The variant TEV proteases can cleave one or more peptides including a TEV protease recognition sequence (e.g., SEQ ID NO:34) with higher efficiency and / or at lower temperatures than, for example, wild-type TEV proteases. Molecules (e.g., proteins) to be cleaved by the variant TEV proteases can include a target protein, a removable fragment, and a TEV protease recognition sequence disposed between the target protein and the removable fragment. The molecules to be cleaved may also include a first linker disposed between the target protein and the TEV protease recognition sequence and / or a second linker disposed between the TEV protease recognition sequence and the removable fragment, wherein the first and second linkers (if both are present) can be identical or different. Examples of molecules to be cleaved can be shown in the N->C direction as follows: Target protein - [adapter-] TEVpRS - [adapter-] removable fragment, or Removable Fragment - [Adapter -] TEVpRS - [Adapter -] Target Protein (collectively referred to as "iB") Short lines represent bonds (covalent or non-covalent), and brackets represent optional elements. In some embodiments, the target protein may be located at the N-terminus or closer to the N-terminus than the removable fragment. According to some embodiments, the target protein may be located at the C-terminus or closer to the C-terminus than the removable fragment. The target protein may be or include one or more of the following: receptors, ligands, enzymes, effectors, antibodies, nucleic acid-binding proteins, structural proteins, hormones, contractile proteins, storage proteins, transport proteins, chaperone proteins, histones, lipoproteins, glycoproteins, toxins, amyloid proteins, variant proteins, misfolded proteins (or any other structurally unique proteins), and / or proteins that bind (covalently or non-covalently) to another molecule (e.g., protein:RNA complex, protein:DNA complex). The removable fragment may be or include one or more of the following: purification tags, affinity tags, fluorophores, solubility tags, epitope tags, expression-enhancing tags, and / or a second target protein.

[0093] The target protein and / or fusion protein comprising the target protein may possess one or more desired properties (e.g., lower catalytic activity, increased stability, improved binding to desired molecules, reduced binding to unwanted molecules), which are enhanced at lower temperatures (e.g., below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C). Therefore, it is desirable to limit or control the conditions of exposure to the target protein and / or fusion protein to maintain the expression of one or more cold-enhanced properties. The provided variant TEV proteases can be used in methods and workflows to perform desired or required cleavage while avoiding the undesirable conditions (e.g., higher temperatures, longer incubation times, higher protease concentrations) that would be obtained using a reference protease (e.g., wild-type TEV protease).

[0094] In some embodiments, a method may include (a) contacting (i) a TEV protease substrate comprising TEVpRS, a target protein (e.g., covalently) attached to the TEVpRS, and a removable fragment (e.g., covalently) attached to a TEV protease recognition sequence (e.g., according to iB) and (ii) a variant TEV protease (e.g., a soluble variant TEV protease or an immobilized (e.g., according to iA) variant TEV protease) to form a cleavage product mixture comprising the target protein and the removable fragment, respectively, and (b) optionally separating the target protein from the removable fragment. Separation of the target protein may be performed by any desired technique, including, for example, affinity chromatography, size chromatography, capillary electrophoresis, filtration, and / or dialysis. Variant TEV proteases may allow the method to be performed at lower temperatures, for example, where the target protein is more stable or has a desired structure or other properties. For example, (a) contact and / or (b) separation can each be carried out independently at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

[0095] In some embodiments, a variant TEV protease can be used in applications involving proteins comprising TEVpRS. For example, the target protein may naturally comprise or be engineered to comprise TEVpRS. Methods of using a variant TEV protease may include contacting the variant TEV protease with a target protein comprising TEVpRS (e.g., at low temperatures) to form a cleavage product (e.g., a cleavage product not containing the complete target protein). For example, it may be desirable to inactivate the enzyme containing TEVpRS after the enzyme-catalyzed reaction is complete, or to prevent the enzyme from becoming active in the presence of a potential substrate. In some embodiments, a method may include contacting an enzyme containing TEVpRS with a substrate at a first temperature (e.g., above 15°C, above 18°C, above 20°C, above 30°C) to produce a reaction product, and contacting the reaction product (or a fragment thereof comprising the enzyme) with the variant TEV protease at a second temperature (e.g., below 10°C, below 5°C) to produce an enzyme cleavage product, wherein the enzyme cleavage product optionally (i) does not contain the complete protein containing TEVpRS and / or (ii) comprises at least two fragments of the protein containing TEVpRS. In some embodiments, the first and second temperatures may optionally be the same. In some implementations, both the target enzyme-catalyzed reaction and the enzyme inactivation reaction can be carried out in a single container (“one-pot”).

[0096] According to some embodiments, a method may include contacting (a) a composition comprising or possibly comprising a protein having TEVpRS (e.g., a target protein) (e.g., a patient sample, cell component, cell extract, or preparation intermediate) with (b) a variant TEV protease (e.g., a soluble variant TEV protease or an immobilized (e.g., according to iA) variant TEV protease) to produce a cleavage product mixture, wherein the cleavage product mixture optionally (i) does not contain an intact protein having TEVpRS and / or (ii) comprises at least two fragments of a protein having TEVpRS, wherein the contact is performed at a temperature below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

[0097] In some embodiments, the enzyme cleavage products and / or mixtures of cleavage products may include little or no intact protein (e.g., target protein) having TEVpRS. For example, the enzyme cleavage products and / or mixtures of cleavage products may include an initial concentration of ≤5 mol%, ≤4 mol%, ≤3 mol%, ≤2 mol%, ≤1 mol%, or ≤0.5 mol% of intact protein (e.g., target protein) having TEVpRS. In some embodiments, ≥95 mol%, ≥96 mol%, ≥97 mol%, ≥98 mol%, ≥99 mol%, ≥99.5 mol%, or ≥99.9 mol% of the protein comprising the TEVpRS substrate may be cleaved by contact with a variant TEV protease, for example, at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

[0098] This disclosure relates to methods for detecting molecular interactions. For example, temperature-sensitive interactions (e.g., protein:protein interactions, enzyme:substrate, enzyme:cofactor, protein:polynucleotide interactions) may occur, may have higher affinity, and / or may have greater affinity at lower temperatures (e.g., temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C). Detection systems that rely on cleavage at higher temperatures (e.g., protein hydrolysis cleavage) may fail to detect such temperature-sensitive interactions (e.g., because cleavage does not occur at all or occurs inefficiently to support detection).

[0099] In some embodiments, a TEV protease substrate may include a TEV protease recognition sequence (“TEVpRS”), a support (e.g., matrix or beads) covalently attached to the TEV protease recognition sequence (e.g., with or without a linker), and a decoy molecule (e.g., protein receptor, ligand, enzyme, effector, antibody, nucleic acid binding protein) covalently attached to the TEV protease recognition sequence (e.g., with or without a linker). A TEV protease substrate can be shown in the N->C direction as follows: Decoy molecule - [connector-]TEVpRS - [connector-] support, or Support material - [Connector-] TEVpRS - [Connector-] Decoy molecule (collectively referred to as "iC") The short lines represent bonds (covalent or non-covalent), and the brackets represent optional elements.

[0100] A method may include (a) contacting (i) a TEV protease substrate comprising TEVpRS, a support (e.g., covalently) attached to a TEV protease recognition sequence, and (e.g., covalently) attached to (e.g., according to iC) a decoy molecule to the TEV protease recognition sequence, and (ii) a composition comprising (or optionally possibly comprising) a target molecule capable of binding the decoy molecule (e.g., with high affinity and / or specificity) to form one or more TEV protease substrate:target molecule complexes (e.g., comprising one or more decoy:target pairs), and (b) contacting one or more TEV protease substrate:target molecule complexes with a variant TEV protease to cleave the TEV protease substrate at the recognition sequence. In some embodiments, cleavage of the TEV protease substrate releases the decoy:target pair from the support. The method may also include analyzing the released decoy:target pair. For example, a method may include analyzing the target member, decoy member, or both members of one or more decoy:target pairs. Variant TEV proteases allow the method to be performed at lower temperatures, for example, to achieve more stable, specific, or otherwise improved bait:target pairing. For example, (a) and / or (b) contact of the method can be performed at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C. In some embodiments, (a) contact can be performed at a first temperature (e.g., above 15°C, above 18°C, above 20°C, above 30°C), and (b) contact can be performed at a second temperature, wherein the second temperature is lower than the first temperature (e.g., below 10°C, below 5°C).

[0101] In some embodiments, ≥95 mol%, ≥96 mol%, ≥97 mol%, ≥98 mol%, ≥99 mol%, ≥99.5 mol%, or ≥99.9 mol% of the TEV protease substrate:target molecule complex are cleaved in the recognition sequence by contact with the variant TEV protease, for example, at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C. In some embodiments, the variant TEV protease can cleave the TEV protease substrate:target molecule complex with higher catalytic efficiency than the control TEV protease. For example, for a given substrate (e.g., TEV protease substrate:target molecule) and a given temperature (e.g., 4°C), the k of the variant TEV protease... cat / K M Compared with the control TEV protease k cat / K M The ratio, for example, can be found in X a To Y b Within the range, where Xa It is any one of 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, or 2.4, and Y b It is any one of 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 and X a <Y b For example, the variant TEV protease versus the control TEV protease kJ cat / K M The ratio can be in the range of 1.3-3.0, 1.4-2.9, 1.5-2.8, or 1.6-2.7.

[0102] Reagent test kit This disclosure also relates to kits that include variant TEV proteases. For example, a kit may include variant TEV proteases, other proteins (e.g., positive and / or negative control substrates), other enzymes, buffers, or combinations thereof. The enzyme may be contained in a storage buffer (e.g., including glycerol and a buffer). The kit may include a reaction buffer, which may be in a concentrated form, and the buffer may contain additives (e.g., glycerol), salts (e.g., KCl), reducing agents, EDTA, or detergents, etc. The kit may be a collection of non-natural components configured, for example, for convenient storage, transport, delivery, and / or use. One or more components of the kit may be contained in a single container for a single-step reaction, or one or more components may be contained in a single container but separated from other components for sequential or parallel use. The contents of the kit may be formulated for use in a desired method or process.

[0103] A kit is provided comprising: (i) a variant TEV protease; and (ii) a buffer. The variant TEV protease may be in any desired form, including, for example, liquid, gel, aqueous, film, crystalline, powder, cake, dry, lyophilized, freeze-dried, and immobilized forms. The variant TEV protease may be contained together with or in a buffer (e.g., a concentrated form of storage buffer or reaction buffer). The kit may contain the variant TEV protease in a master mixture suitable for receiving and amplifying target template nucleic acids. The variant TEV protease may be a purified enzyme, thus substantially free of DNA, RNA, nucleases, and / or other proteases. The reaction buffer in (ii) and / or the storage buffer containing or containing the TEV protease in (i) may include nonionic, ionic (e.g., anionic or zwitterionic) surfactants and / or aggregation agents. The kit may include the variant TEV protease and reaction buffer in a single container or in separate containers.

[0104] The kit may also include instructions on how to practice the required methods using the kit's components. These instructions may be recorded on a suitable recording medium. For example, they may be printed on a substrate such as paper or plastic. Therefore, the instructions may be present as a packaging insert within the kit, or on the label of the container of the kit or its components (i.e., associated with the packaging or sub-packaging), etc. The instructions may also exist as an electronic storage data file residing on a suitable computer-readable storage medium (e.g., CD-ROM, flash drive). For example, the instructions may be provided remotely via a link using resources such as the cloud or the internet, or other accessible instructions may be provided within or with the kit.

[0105] Example Some specific implementation examples can be illustrated through one or more embodiments provided herein.

[0106] Example 1: Synthesis of TEV protease variants A codon-optimized TEV protease gene with L56V, S135G, and S219V, along with an N-terminal His tag (SEQ ID NO:30), was cloned into the pUCT7 plasmid, a pUC19-based plasmid with a T7 promoter, followed by a Shine-Dalgarno sequence upstream of the open reading frame (ORF) and a T7 terminator sequence downstream of the ORF. L56V and S135G substitutions were included to improve solubility, and S219V was included to prevent self-hydrolysis. A TEV protease with these three substitutions (“TEV protease triple variant” or “control”) was used as a reference in this study. Other substitutions added to the triple variant sequence of the TEV protease include one or more of K45A, K45G, K45H, K45Q, K45R, K45S, R49E, R49K, R49V, I77C, I77L, I77V, D78E, D78G, D78K, D78Q, M82A, M82I, M82L, M82V, I83L, I83M, I83V, I84L, I84V, Q150D, Q150F, Q150H, S153L, S153N, L155A, L155I, L155M, and L155V, wherein the substitutions are numbered according to the amino acid position of the wild-type TEV protease (SEQ ID NO:31).

[0107] Plasmids carrying TEV protease variants were constructed using the Q5 site-directed mutagenesis kit (New England Biolabs). TEV protease variants were synthesized in vitro using a modified version of PURExpress (New England Biolabs). 100–200 ng of plasmid and 20 units of the RNase inhibitor Murine (New England Biolabs) were added to 25 µl of the modified PURExpress reaction and incubated at 37 °C for 3 h. The reaction mixture was stored at -20 °C for further analysis.

[0108] Optionally, the N-terminal histidine-tagged TEV protease variant was expressed in vivo as a maltose-binding protein fusion. The TEV protease cleavage site located between the MBP and the variant sequence allows for intracellular cleavage of the fusion protein. Cells were lysed and the N-terminus was cleaved, and the His-tagged TEV protease variant was purified by chromatography. Transformed T7 Express competent cells were grown in 1 L LB medium at 37°C until the OD reached 0.8 A. 600 Then, 1 mL of 0.5 M IPTG was added, and the culture was incubated overnight at 18°C. Cells were collected and resuspended in 50 mL of Tris buffer (pH 7.5). The suspended cells were lysed using a microfluidic apparatus (Dyhydromatics) and centrifuged twice at 15,000 rpm for 20 minutes to remove cell debris. The supernatant was loaded onto a HisTrap FF 5 mL (GE Healthcare) and eluted with a gradient of Tris buffer (pH 7.5) containing imidazole. The eluted protein was collected and dialyzed overnight at 4°C with storage buffer containing reducing agent and 50% glycerol. Protein concentration was determined using the Bradford assay. The purified protein was stored at -80°C. Results of the examples are as follows. Figure 2 As shown.

[0109] Example 2: Quantification of TEV protease variants in in vitro synthesis reaction 2.5 µl of the in vitro synthesis reaction solution was mixed with 7.5 µl of H₂O and 5 µl of 3x SDS sample buffer (New England Biolabs). The solution was incubated at 95 °C for 5 min, rotated at 14,000 rpm for 1 min, and then loaded into Novex. TM WedgeWell TM Loaded onto 4-20% Tris-Glycine gels (Invitrogen). Plasmid-free reactants were loaded as negative controls. Unstained protein standards (New England Biolabs) with a wide range (10-200 kDa) were used as labels. SimplyBlue was used.TM The gel was stained with SafeStain (Life Technologies), and the TEV protease variant bands were quantified using the Odyssey imaging system (LI-COR Biosciences). The concentration of the TEV protease variant relative to the control TEV protease was then calculated.

[0110] Example 3: Fluorescent peptide cleavage assay of TEV protease variants The fluorescent peptide (5-FAM-ENLYQGIV-K(QXL520)-NH2; SEQ ID NO:32) was used as a substrate (AnaSpec). It is 9 amino acids long and includes a TEV protease recognition sequence (SEQ ID NO:34). The fluorophore 5-FAM is attached to the N-terminus, and the quencher QXL520 is attached to the ε-amino group of the C-terminal lysine residue. The peptide exhibits fluorescence if cleaved by the TEV protease. The excitation and emission wavelengths are 493 nm and 517 nm, respectively.

[0111] In a test tube, mix 2.5 µl of 10x TEV protease reaction buffer (500 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 10 mM DTT) (New England Biolabs), 2.5 µl of 100 µM peptide substrate, and 19 µl of H2O. Initiate the reaction by adding 1 µl of in vitro synthesis reaction mixture diluted with H2O. Each reaction was incubated at 15 °C for 30 min, followed by heat inactivation at 65 °C for 10 min using a T100 thermal cycler (Bio-Rad). Aliquots of the inactivated reaction mixture were added to the wells of a 384-well plate (10 µL mixture / well), and fluorescence was measured using a SpectraMax M5 (Molecular Devices). Four measurements were performed, and the average value was calculated.

[0112] Example 4: CBD-TEVrs-bglA Cutting Test CBD-TEVrs-bglA was prepared as a substrate for purifying the TEV protease variant. This protein consists of an N-terminal His-tagged chitin-binding domain (CBD), followed by TEVrs (ENLYFQG) and 6-phosphate-β-glucosidase A (bglA) (SEQ ID NO:33). The CBD-TEVrs-bglA gene was cloned into the pET28 vector. CBD-TEVrs-bglA was expressed in vivo and purified using a nickel column in the same manner as the aforementioned TEV protease variant.

[0113] Mix 5 µl of 10x TEV protease reaction buffer, 5 µl of 370 µM CBD-TEVrs-bglA, and 38 µl of H2O in a test tube. Initiate the lysis reaction by adding 2 µl of 10 µM TEV protease to the test tube and incubate the reaction tube at a selected temperature for a selected time using a thermal cycler (0 °C 18 h, 4 °C 18 h, 20 °C 3 h, or 30 °C 2 h). At intervals, remove aliquots (5 µl each) (every 30 min at 20 °C and every 20 min at 30 °C), mix with cold acetone (50 µl), and keep on ice until all intervals of the given treatment have been collected.

[0114] Rotate the test tube at 14,000 rpm for 10 minutes at 4°C. Then discard the supernatant and allow the precipitate to dry at room temperature for 10 minutes. Dissolve the precipitate in 40 µl of H₂O and 20 µl of 3x SDS sample buffer (New England Biolabs). Incubate the solution in the test tube at 95°C for 5 minutes and rotate at 14,000 rpm for 1 minute. Then load 10 µl of the solution (2 µg of CBD-TEVrs-bglA) into Novex. TM WedgeWell TM Apply 4-20% Tris-Glycine gel (Invitrogen). Use SimplyBlue. TM SafeStain (Life Technologies) stained the gel and used the Odyssey imaging system (LI-COR Biosciences) to quantify the bands of CBD-TEVrs-bglA and bglA, from which the cleavage ratio of CBD-TEVrs-bg1A was calculated.

[0115] Example 5: Titration with control and TEV protease variant The control or TEV protease variant stock solution (50 µM) was serially diluted 2-fold with storage buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 50% glycerol). In a test tube, mix 5 µL of 10x TEV protease reaction buffer, 5 µL of 370 µM CBD-TEVrs-bglA, and 38 µL of H2O. Initiate the cleavage reaction by adding 2 µL of diluted TEV protease to the test tube and incubate at 4 °C for 18 h. Remove an aliquot (5 µL) from each test tube and mix with 50 µL of cold acetone. Rotate the test tube at 14,000 rpm for 10 min at 4 °C. Then discard the supernatant and allow the precipitate to dry at room temperature for 10 min. Dissolve the precipitate in 40 µL of H2O and 20 µL of 3x SDS sample buffer (New England Biolabs). The solution in the test tube was incubated at 95°C for 5 minutes, rotated at 14000 rpm for 1 minute, and then 10 µl of solution (2 µg CBD-TEVrs-bglA) was loaded into Novex. TM WedgeWell TM Apply 4-20% Tris-Glycine gel (Invitrogen). Use SimplyBlue. TM SafeStain (Life Technologies) stained the gel and used the Odyssey imaging system (LI-COR Biosciences) to quantify the bands of CBD-TEVrs-bglA and bglA, from which the cleavage ratio of CBD-TEVrs-bg1A was calculated.

[0116] Example 6: Identification of Fluorescent Peptide Cleavage Reaction Conditions Prior to the fluorescence assay used in Example 3, the product from the in vitro synthesis reaction was diluted with water so that the fluorescence intensity was proportional to the activity of the TEV protease. To determine the appropriate dilution level, the correlation between the concentration of the control TEV protease and the fluorescence was measured after incubation at 15°C for 30 minutes. Figure 1 A good linear correlation was observed when the control TEV protease was diluted more than 4-fold. Therefore, in the first round of screening, the control TEV protease was diluted 5-fold with water. Since enzyme activity depends on enzyme concentration, it is crucial to add the same amount of the TEV protease variant as the control TEV protease in the fluorescent peptide cleavage reaction. The concentration of the TEV protease variant was diluted with water to match the concentration of the 5-fold diluted control TEV protease.

[0117] Example 7: Analysis of proline variants produced in vitro As described in Example 1, three further proline-substituted TEV protease triple variants were synthesized in vitro: K67P, Q73P, L76P (SEQ ID NO:6); D78P, K89P (SEQ ID NO:7); E102P, S181P (SEQ ID NO:8); and K184P, Q193P, A206P, G213P, or E223P (according to SEQ ID NO:31). The in vitro synthesized products were evaluated on an SDS gel along with a control TEV protease (SEQ ID NO:29), as described in Example 2. Results are as follows: Figure 2 As shown. Some changes in protein synthesis were noted in the variants. The TEV protease variant bands were quantified on an SDS gel, and the concentration of the TEV protease variant was adjusted to match the concentration of the control TEV protease.

[0118] According to Example 3, the cleavage reaction of the fluorescent peptide substrate with the TEV protease variant was carried out at 15°C for 30 minutes. The results are as follows... Figure 3 As shown. Among the 12 TEV protease variants, the L76P, K89P, and S181P variants exhibited higher activity than the control TEV protease (SEQ ID NO:29).

[0119] Example 8: Analysis of in vitro generated catalytic triple variants As described in Example 1, a TEV protease triple variant (SEQ ID NO:29) was synthesized in vitro, comprising further substitutions (K45A, K45G, K45H, K45Q, K45R, K45S, R49E, R49K, R49V, I77C, I77L, I77V, D78E, D78G, D78K, D78Q, M82A, M82I, M82L, M82V, I83L, I83M, I83V, I84L, I84V, Q150D, Q150F, Q150H, S153L, S153N, L155A, L155I, L155M, or L155V, according to SEQ ID NO:31) in or near the predicted catalytic triplet (H46, D81, and C151, according to SEQ ID NO:31). (ID NO: 31). The synthesized product was fractionated on an SDS gel, and the TEV protease variant and control bands were quantified as described in Example 2. The concentration of the TEV protease variant was adjusted to match the concentration of the control TEV protease.

[0120] Nine substitutions were introduced near position H45, and assays were performed at 15°C for 30 minutes according to Example 3. Of these nine, K45Q, K45R, and R49V showed higher activity than the control TEV protease (SEQ ID NO:29). Figure 4Sixteen substitutions were introduced near D81 and nine near C151. Some of these substitutions exhibited catalytic activity, but none of them exceeded the activity of the control TEV protease under the test conditions.

[0121] Example 9: Analysis of in vitro generated combinatorial variants As described in Example 1, a TEV protease triple variant (SEQ ID NO: 29) comprising further substitutions of K45R, R49V, L76P, K45R_R49V, K45R_L76P, or R49V_L76P (according to SEQ ID NO: 31) was synthesized in vitro, the concentration was adjusted, and the assay was performed at 15°C for 30 minutes according to Example 3. Results are as follows: Figure 5 As shown. Further TEV protease triple variants including K45R, R49V, K45R_R49V, or K45R_L76P each exhibited activity comparable to the control. Variants with R49V_L76P substitution (SEQ ID NO:3) and variants with L76P substitution (SEQ ID NO:6) exhibited activity exceeding that of the control (~1.4x - ~1.5x).

[0122] Example 10: Analysis of variants produced in vivo The TEV protease variant of Example 9 was prepared in vivo according to Example 1. The cleavage reaction of CBD-TEVrs-bglA with the purified TEV protease variant was evaluated over time at 30°C and 20°C according to Example 4. The results are as follows... Figure 6 (30℃) and Figure 7 As shown at (20℃). The R49V variant exhibited slightly weaker activity than the control TEV protease (SEQ ID NO:30). At both temperatures, the L76P variant showed higher activity than the control TEV protease, and the R49V_L76P variant showed higher activity than the L76P variant.

[0123] The cleavage of CBD-TEVrs-bglA by these TEV protease variants was assessed at 4°C for 18 hours. Results are as follows: Figure 8 As shown, the TEV protease variants exhibited greater cleavage rates at 4°C than at 20°C. The control TEV protease cleaved 73% of the substrate, while the R49V_L76P variant cleaved 94%, indicating that the R49V_L76P variant can cleave protein substrates at 4°C and has higher activity than the control TEV protease or variants containing only one of the R49V or L76P substitutions.

[0124] Example 11: Analysis of Catalytic Triad Variants Based on the crystal structure data of the TEV protease-substrate complex, amino acids R49 and L76 do not bind to the substrate, but they are located near the catalytic triplet. As described in Example 1, a TEV protease triple variant comprising one of 11 R49X_L76P substitutions was expressed in vitro, concentrations were adjusted, and the expression was evaluated at 10°C for 60 minutes according to Example 3. Figure 9 As shown, the hydrophobic amino acid substitution at R49 is associated with increased activity. Variants marked with arrows (R49A_L76P, R49F_L76P (SEQ ID NO:11), R49G_L76P, R49I_L76P, R49L_L76P, and R49V_L76P (SEQ ID NO:12)) were expressed in vivo and evaluated at 30°C according to Example 4. Figure 10 As shown, all variants outperformed the control, with the R49F_L76P variant (SEQ ID NO:11) exhibiting the highest activity.

[0125] Example 12: Low-temperature performance of variant TEV protease According to Example 4, the cleavage of CBD-TEVrs-bglA by the L76P variant (SEQ ID NO:15), R49F_L76P variant (SEQ ID NO:11), and control variant protease (SEQ ID NO:30) was evaluated at 0°C or 4°C for 18 hours. Results are as follows: Figure 12 As shown. Variants L76P and R49F_L76P exhibited approximately 10% and 20% higher activities than the control TEV protease, respectively, at both temperatures. At a assay temperature of 4°C, the control TEV protease cleaved 77% of the substrate, while the L76P variant cleaved 87% (10% more than the control), and the R49F_L76P variant cleaved 97% (20% more than the control). Similarly, at a assay temperature of 0°C, the control TEV protease cleaved 64% of the substrate, while the L76P variant cleaved 75% (11% more than the control), and the R49F_L76P variant cleaved 86% (22% more than the control).

[0126] Example 13: Kinetic Analysis of Catalytic Triad Variants The R49V variant (SEQ ID NO:14), L76P variant (SEQ ID NO:15), R49V_L76P variant (SEQ ID NO:12), and R49F_L76P variant (SEQ ID NO:11) were selected for further analysis. According to Example 5, the cleavage activity of CBD-TEVrs-bglA was measured as a function of TEV protease concentration (2-fold serial dilution) at 4°C for 18 hours.

[0127] like Figure 13CAs shown, the R49V_L76P variant achieved almost 100% cleavage at 0.5 µM, while the control TEV protease required 2 µM to achieve the same effect. Figure 13D As shown, the R49F_L76P variant achieved almost 100% cleavage at 0.5µM, while the control TEV protease required 2µM to achieve the same effect.

[0128] Kinetic parameters were calculated using data from the linear phase of the reaction. The natural logarithm of (1 - (cleavage ratio)) versus TEV protease concentration was plotted, and the slope value k was calculated, as shown in Table 2-5. Figures 14A-14D The calculated slope difference between the tested variant and the control is shown, indicating that the tested variant was more active than the control under the test conditions. For example, Figure 14A and Figure 14B The results showed that the R49V variant had 2.0 times the activity of the control, and the L76P variant had 1.7 times the activity of the control. Figure 14C The results show that the R49V_L76P variant exhibits 1.5 times the activity of the control TEV protease. Figure 14D As shown, the slope difference between the R49F_L76P variant and the control TEV protease was 2.6-fold. This result indicates that, at 4°C, the activity of the R49F_L76P variant is 2.6 times that of the control TEV protease.

[0129] Example 14: Comparison with commercial enzymes The activities of the R49F_L76P variant TEV protease generated in vivo according to Example 1 and a commercially available TEV protease (New England Biolabs, catalog number #P8112S) on the example substrate MBP-TEV-paramyosin ΔSal (SEQ ID NO:35) were compared at 4°C. Cleavage of MBP-TEV-paramyosin ΔSal (70.7 kDa) at the TEV protease recognition site yielded two products, MBP (43.7 kDa) and paramyosin ΔSal (27.3 kDa).

[0130] Activity assays were performed using 10 µg of MBP-TEV-paramyosin ΔSal mixed with 0.625 µg of TEVp in 1x TEV protease reaction buffer (50 µL final volume). A negative control (substrate + reaction buffer, enzyme-free) and two enzyme reactions (0.125 µg enzyme per reaction per assay) were prepared on ice and incubated at 4 °C for 5 h. Aliquots (10 µL each) were harvested at start (T0), 30 min (T0.5), 1 h (T1), 3 h (T3), and 5 h (T5), incubated at 65 °C for 10 min to inactivate the enzyme, and combined with 5 µL of 3x SDS sample buffer (New England Biolabs). These volumes were then incubated at 95 °C for 2 min followed by rotation at 14,000 rpm for 1 min. 10 µL of each was then loaded into Novex. TM WedgeWell TM Apply to 4-20% Tris-Glycine gel (Invitrogen). After gel run, use SimplyBlue. TM SafeStain (Life Technologies) staining was performed, and the bands of MBP-TEV-paramyosin ΔSal MBP, paramyosin ΔSal and MBP were quantified using the Odyssey imaging system (LI-CORBiosciences) to calculate the cleavage ratio of MBP-TEV-paramyosin ΔSal.

[0131] The results are as follows Figure 15 As shown in the diagram. Under the test conditions, the variant protease outperformed the commercially available enzyme at every time point. The variant protease achieved 85% cleavage in just 1 hour at 4°C, while the commercially available enzyme only achieved 62% cleavage in the same time period. The variant TEV protease cleaved 98% of the substrate in 3 hours and 99% of the substrate in 5 hours, while the commercially available enzyme only cleaved 76% and 83% of the substrate, respectively, at these times.

Claims

1. A cold-active variant of the TEV protease having an amino acid sequence that is at least 90% identical to that of SEQ ID NO:1, wherein X49 is any amino acid except arginine, X73 is any amino acid except glutamine, X76 is any amino acid except leucine, X78 is any amino acid except aspartic acid, X82 is any amino acid except methionine, X83 is any amino acid except isoleucine, X84 is any amino acid except isoleucine, X89 is any amino acid except lysine, X102 is any amino acid except glutamic acid, X150 is any amino acid except glutamine, X155 is any amino acid except leucine, X181 is any amino acid except serine, X184 is any amino acid except lysine, X193 is any amino acid except glutamine, X206 is any amino acid except alanine, or X223 is any amino acid except glutamic acid.

2. A cold-active variant of the TEV protease having an amino acid sequence that is at least 90% identical to that of SEQ ID NO:1, wherein at least one of X49, X73, X76, X78, X82, X83, X84, X89, X102, X150, X155, X181, X184, X193, X206 and X223 constitutes a substitution relative to the corresponding position of SEQ ID NO:

31.

3. The cold-active variant TEV protease according to claim 1 or claim 2, having an amino acid sequence that is at least 98% identical to SEQ ID NO:2 or 3 or at least 97% identical to SEQ ID NO:20 or 21.

4. The cold-active variant TEV protease according to claim 1 or claim 2, wherein (a) X49 is any amino acid other than arginine, or (b) X76 is any amino acid other than leucine, or (c) X49 is any amino acid other than arginine and X76 is any amino acid other than leucine.

5. The cold-active variant TEV protease according to any of the preceding claims, wherein (a) X49 is phenylalanine or valine, or (b) X76 is proline, or (c) X49 is phenylalanine or valine and X76 is proline.

6. The cold-active variant TEV protease according to any of the preceding claims, wherein the amino acid sequence includes one or more of the following substitutions: 45-position substitution, 56-position substitution, 135-position substitution, and 219-position substitution.

7. The cold-active variant TEV protease according to any of the preceding claims, wherein the amino acid sequence comprises one or more of K45Q substitution, K45R substitution, L56V substitution, S135G substitution, and S219V substitution.

8. The cold-active variant TEV protease according to any of the preceding claims, wherein the amino acid sequence corresponds to SEQ ID NO:1 at positions 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 141, 146, 147, 148, 15 One or more of the following are identical: 1, 153, 158, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, and 237.

9. A cold-active variant of the TEV protease, comprising an amino acid sequence characterized by: (a) It shares at least 90% identity with SEQ ID NO:

1. (b) At least one substitution relative to SEQ ID NO:31 at positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 corresponding to positions SEQ ID NO:

31. (c) Optionally, it may be replaced at any position or any combination of positions 56, 135 and 219 corresponding to SEQ ID NO:31, and (d) Optionally, at positions 2, 3, 12, 17, 23, 28, 30, 45, 50, 56, 58, 67, 68, 74, 77, 79, 80, 87, 90, 93, 106, 107, 110, 119, 120, 122, 127, 130, 132, 135, 138, 141, 146, 147, 148, 151, 153, 1 corresponding to SEQ ID NO:31 At any position or any combination of positions 58, 162, 168, 170, 171, 173, 176, 177, 180, 203, 205, 209, 211, 215, 216, 217, 218, 219, 220, 224, 225, 226, 227, 228, 229, 235, 236, 237, the protease is identical to that of wild-type TEV at any position or combination of positions.

10. A cold-active variant of the TEV protease, comprising the following amino acid sequence: (a) Has at least 90% identity with SEQ ID NO:1, (b) Having at least one substitution relative to wild-type TEV protease (SEQ ID NO:31) at positions 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, and 223 corresponding to SEQ ID NO:

31. (c) Optionally having a substitution at any position or any combination of positions 56, 135 and 219 corresponding to SEQ ID NO:31, (d) Optionally, E2K, S3P, N12D, T17S, N23Q, H28L, H28Y, T30A, T30I, K45F, K45W, R50G, L56V, Q58F, Q58Y, Q58I, K67E, N68D, Q74L, I77V, G79E, R80S, M87L, M87T, D90G, P93S, E106G, E107D, C110S corresponding to SEQ ID NO:31 , K119E, S120R, S122P, D127A, C130S, F132L, F132S, S135G, S135F, I138T, K141R, T146A, T14 6C, T146S, K147E, D148R, D148P, D148A, C151A, S153C, S153N, T158A, F162S, F162A, S168T, S 170A, N171D, N171Q, T173A, T173G, N176I, N176T, N177K, N177R, N177S, N177M, N177Y, T180 A. R203Q, N205D, V209M, V209F, W211I, W211V, W211L, W211C, K215E, V216I, F217K, M218I, M2 One or more of the above substitutions are missing at the positions of 18F, M218W, M218L, M218T, S219D, S219E, S219V, S219P, S219N, K220R, P224S, F225L, Q226stop, Q226S, Q226P, P227A, V228S, K229E, K229stop, M235K, N236S, and E237G; and (e) Optionally, one or more of the above substitutions are omitted at the positions corresponding to K67P, M82I, I83V, Q150D, Q150H, S153L, L155A, L155M and G213P of SEQ ID NO:

31.

11. A cold-active variant of the TEV protease having an amino acid sequence that is at least 98% identical to SEQ ID NO:9 or 10, at least 97% identical to SEQ ID NO:27 or 28, or at least 95% identical to SEQ ID NO:18 or 19.

12. The cold-active variant TEV protease according to any of the preceding claims, wherein the k of the variant TEV protease is... cat / K M Compared with the control TEV protease k cat / K M The ratio is in the range of 1.3-3.

0.

13. A method comprising: (a) to cause: (i) A TEV protease substrate comprising a TEV protease recognition sequence having the amino acid sequence of SEQ ID NO:34, a target protein attached to the TEV protease recognition sequence, and a removable fragment attached to the TEV protease recognition sequence. (ii) a variant TEV protease contact according to any one of claims 1-12 to form a mixture of cleavage products comprising the target protein and the removable fragment, respectively; and (b) Optionally, the target protein is separated from the removable fragment. The contact is carried out at a temperature below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

14. A method comprising: make: (a) A composition comprising or possibly comprising a protein, said protein comprising a TEV protease recognition sequence having the amino acid sequence SEQ ID NO:34, and (b) Contact with the variant TEV protease according to any one of claims 1-12 to produce a mixture of cleavage products. The cutting product mixture is optionally (i) Does not contain a complete protein including the TEV protease recognition sequence, and / or (ii) Contains at least two fragments of a protein including the TEV protease recognition sequence, and The contact is carried out at a temperature below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

15. A method comprising: (a) to cause: (i) A TEV protease substrate comprising a TEV protease recognition sequence having the amino acid sequence of SEQ ID NO:34, a support attached to the TEV protease recognition sequence, and a decoy molecule attached to the TEV protease recognition sequence, and (ii) Composition contact comprising a target molecule capable of binding the decoy molecule to form one or more TEV protease substrate:target molecule complexes, and (b) Contacting the one or more TEV protease substrate:target molecule complexes with a variant TEV protease according to any one of claims 1-12 to cleave the TEV protease substrate at the recognition sequence. (a) contact, (b) contact, or (a) contact and (b) contact are performed at temperatures below 20°C, below 18°C, below 16°C, below 14°C, below 12°C, below 10°C, below 8°C, below 6°C, below 4°C, below 2°C, or below 0°C.

16. The method of claim 13, 14, or 15, wherein the k of the variant TEV protease cat / K M Compared with the control TEV protease k cat / K M The ratio is in the range of 1.3-3.

0.

17. The method of claim 13, 14, or 15, wherein the variant TEV protease is a soluble variant TEV protease or an immobilized variant TEV protease.

18. A reagent kit comprising: (a) The variant TEV protease according to any one of claims 1-12; and (b) Buffer solution The variant TEV protease is selected from liquid, gel, aqueous, film, crystalline, powder, cake, dry, freeze-dried, lyophilized and immobilized forms, and the buffer is a reaction buffer or a storage buffer.

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