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
pTXB1
pTXB3
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
pTYB21
pTYB22
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
pTYB1
pTYB2
pTYB3
pTYB4
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)
pH 8.0-8.5, 4°C
Purification and ligation
pTWIN1 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
pTWIN2 C-terminus
(Intein 2)
Mth RIR1
intein (22)
NdeI-SapI (or SpeI) G, A, LEG
(Unfavorable residues- P, E, D)
pTWIN1d 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
pTWIN1d 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
pTWIN2d 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.