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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 |
Applications |
| pTYB1 pTYB2 pTYB3 pTYB4 |
C-terminus | Sce VMA1 intein (56) |
Nde I-Sap I Nde I-Sma I (or Xho I) Nco I-Sap I Nco I-Sma I (or Xho I) |
G, LEG (Unfavorable residues- P, C, N, D, R) |
DTT (or MESNA) pH 8.0-8.5, 4°C |
Purification and ligation |
| pTXB1 pTXB3 |
C-terminus | Mxe GyrA intein (28) |
Nde I-Sap I (or Spe I) | M, Y, F, LEM (Unfavorable residues- S, P, E, D, A) |
Purification; C-terminal thioester for ligation and modification |
|
| pTWIN1 | C-terminus (Intein 2) |
Mxe GyrA intein (28) |
Nde I-Sap I (or Spe I) | M, Y, F, LEM (Unfavorable residues- S, P, E, D, A) |
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| pTWIN2 | C-terminus (Intein 2) |
Mth RIR1 intein (22) |
Nde I-Sap I (or Spe I) | G, A, F, LEG (Unfavorable residues- P, E, D) |
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| pTWIN1 | N-terminus (Intein 1) |
Ssp DnaB mini-intein (27) |
Sap I-Sap I Sap I-Pst I (or BamH I) BsrG I-Pst I (or BamH I) |
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 |
| pTYB11 pTYB12 |
N-terminus | Sce VMA1 intein (56) |
Sap I-Pst I Bsm I (or Nde I)-Not I |
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 |
| pTWIN1 | N-terminus (Intein 1) & C-terminus (Intein 2) |
Ssp DnaB mini-intein (27) Mxe GyrA intein (28) |
Sap I-Sap I | C, S, A, G, M, T, CRAM (Unfavorable residue- P) M, Y, F, LEM (Unfavorable residues- S, P, E, D, A) |
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 Cyclizationd |
| pTWIN2 | N-terminus (Intein 1) & C-terminus (Intein 2) |
Ssp DnaB mini-intein (27) Mth RIR1 intein (22) |
Sap I-Sap I | C, S, A, G, M, T, CRAM (Unfavorable residue- P) G, A, LEG (Unfavorable residues- P, E, D) |
aActual cleavage efficiency is dependent on the adjacent residues as well as the folding of the fusion
protein.
bDithiothreitol (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.
cCysteine can be used in the place of DTT.
dWhen creating circular proteins it is
typical that the linear form will be co-purified, requiring a further step to separate the two
protein species.