Cold-active tev proteases, compositions and methods
AssigneeNew England Biolabs, Inc.
InventorsHaruichi Asahara, James C. Samuelson
Life of this patent
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Abstract
The present disclosure relates, according to some embodiments, to compositions, methods and kits for cleaving a target protein using a cold-active variant TEV protease.
Claims22
A cold-active variant TEV protease having an amino acid sequence at least 90% identical to SEQ ID NO:1, wherein 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 aspartate, 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 glutamate, 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, or X223 is any amino acid other than glutamate.
A cold-active variant TEV protease having an amino acid sequence 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 of SEQ ID NO:31.
A cold-active variant TEV protease comprising an amino acid sequence having:
- (a) at least 90% identity to SEQ ID NO:1,
- (b) at least one substitution relative to SEQ ID NO:31 at a position corresponding to position 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, 223 of SEQ ID NO:31,
- (c) optionally a substitution at any of its positions or any combination of its positions corresponding to positions 56, 135, and 219 of SEQ ID NO:31, and
- (d) optionally identity to wildtype TEV protease at any of its positions or any combination of its positions corresponding to 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, 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, 237 of SEQ ID NO:31.
A cold-active variant TEV protease comprising an amino acid sequence:
- (a) having at least 90% identity to SEQ ID NO:1,
- (b) having at least one substitution relative to wildtype TEV protease (SEQ ID NO:
- 31) at a position corresponding to position 49, 73, 76, 78, 82, 83, 84, 89, 102, 150, 155, 181, 184, 193, 206, 223 of SEQ ID NO:31,
- (c) optionally having a substitution at any of its positions or any combination of its positions corresponding to positions 56, 135, and 219 of SEQ ID NO:
- 31,
- (d) optionally lacking one or more of the following substitutions at its positions corresponding to:
- 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, 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, N171Q, T173A, T173G, N176I, N176T, N177K, N177R, N177S, N177M, N177Y, T180A, R203Q, N205D, V209M, V209F, W211I, W211V, W211L, W211C, K215E, V216I, F217K, M218I, M218F, M218W, M218L, M218T, S219D, S219E, S219V, S219P, S219N, K220R, P224S, F225L, Q226stop, Q226S, Q226P, P227A, V228S, K229E, K229stop, M235K, N236S, and E237G of SEQ ID NO:
- 31;
- and
- (e) optionally lacking one or more of the following substitutions at its positions corresponding to:
- K67P, M82I, 183V, Q150D, Q150H, S153L, L155A, L155M, and G213P of SEQ ID NO:31.
A cold-active variant TEV protease having an amino acid sequence at least 98% identical to SEQ ID NO:9 or 10 or at least 97% identical to SEQ ID NO:27 or 28 or at least 95% identical to SEQ ID NO:18 or 19.
Dependencies are inferred from the claim text.