NEB IMPACT
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IMPACT Vectors and Applications
| Vectors | Site of Target Protein Fusion |
Intein Tag (kDa) |
Recommended Cloning Sites |
Preferred Residues at Cleavage Sitea | Method of Cleavageb,c | Applications |
| C-terminus | Mxe GyrA intein (28) |
NdeI-SapI (or SpeI) | Y, F, Q, N, T, K, A, H, M, LEM (Unfavorable residues- S, P, D, G) |
DTT (or MESNA) pH 8.0-8.5, 4°C |
Purification; C-terminal thioester for ligation and modification | |
| N-terminus | Sce VMA1 intein (56) |
SapI-PstI BsmI (or NdeI)-PstI |
A, Q, M, G, L, N, W, F, Y (Unfavorable residues- P, S, C, T, R) |
DTTc pH 8.0-8.5, 25°C |
Purification | |
| C-terminus | Sce VMA1 intein (56) |
NdeI-SapI NdeI-SmaI (or XhoI) NcoI-SapI NcoI-SmaI (or XhoI) |
G, A, M, F, Q, W, Y, K, LEG (Unfavorable residues- P, C, N, D, R) |
DTT (or MESNA) |
Purification and ligation | |
| C-terminus (Intein 2) |
Mxe GyrA intein (28) |
NdeI-SapI (or SpeI) | Y, F, Q, N, T, K, A, H, M, LEM (Unfavorable residues- S, P, D, G) |
Purification; C-terminal thioester for ligation and modification | ||
| C-terminus (Intein 2) |
Mth RIR1 intein (22) |
NdeI-SapI (or SpeI) | G, A, LEG (Unfavorable residues- P, E, D) | |||
| N-terminus (Intein 1) |
Ssp DnaB mini-intein (27) |
SapI-SapI SapI-PstI (or BamHI) BsrGI-PstI (or BamHI) |
C, S, A, G, M, T, CRAM (Unfavorable residue- P) |
pH 6.0-7.0, 25°C |
Purification; Defined N-terminus (e.g. Cys); Ligation | |
| N-terminus & (Intein 1) C-terminus (Intein 2) |
Ssp DnaB mini-intein (27) Mxe GyrA intein (28) |
SapI-SapI | C, S, A, G, M, T, CRAM (Unfavorable residue- P) Y, F, Q, N, T, K, A, H, M, LEM (Unfavorable residues- S, P, D, G) |
Step 1: pH 6.0-7.0, 25°C Step 2: DTT (or MESNA) pH 8.0-8.5, 4°C |
Purification; Defined N-terminus (e.g. Cys); Ligation and Cyclizatione | |
| N-terminus & (Intein 1) C-terminus (Intein 2) |
Ssp DnaB mini-intein (27) Mth RIR1 intein (22) |
SapI-SapI | C, S, A, G, M, T, CRAM (Unfavorable residue- P) G, A, LEG (Unfavorable residues- P, E, D) |
a Actual cleavage efficiency is dependent on the adjacent residues as well as the folding of the fusion protein.
b Dithiothreitol (DTT) is used only for protein purification. 2-mercaptoethanesulfonic acid (MESNA) is used for isolation of proteins possessing a C-terminal thioester for ligation, labeling and cyclization.
c Cysteine can be used in the place of DTT.
d pTWIN vectors allow for induction of intein cleavage by a pH/temperature shift.
e When creating circular proteins it is typical that the linear form will be co-purified, requiring a further step to separate the two protein species.