| Vectors |
Site of Target
Protein Fusion |
Intein Tag
(kDa) |
Recommended
Cloning Sites |
Preferred Residues
at Cleavage Sitea |
Method of
Cleavageb,c |
Applications |
pTYB1
pTYB2
pTYB3
pTYB4 |
C-terminus |
Sce VMA1
intein (56) |
NdeI-SapI
NdeI-SmaI (or XhoI)
NcoI-SapI
NcoI-SmaI (or XhoI) |
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) |
NdeI-SapI (or SpeI) |
M, Y, F, LEM
(Unfavorable residues- S, P, E, D) |
Purification; C-terminal
thioester for ligation and modification |
| pTWIN1 |
C-terminus
(Intein 2) |
Mxe GyrA
intein (28) |
NdeI-SapI (or SpeI) |
M, Y, F, LEM
(Unfavorable residues- S, P, E, D) |
| pTWIN2 |
C-terminus
(Intein 2) |
Mth RIR1
intein (22) |
NdeI-SapI (or SpeI) |
G, A, LEG
(Unfavorable residues- P, E, D) |
pTYB11
pTYB12 |
N-terminus |
Sce VMA1
intein (56) |
SapI-PstI
BsmI (or NdeI)-NotI |
A, Q, M, G, L, N, W, F, Y
(Unfavorable residues- P, S, C, T, R) |
DTT c
pH 8.0-8.5, 25°C |
Purification |
| 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)
M, Y, F, LEM
(Unfavorable residues- S, P, E, D) |
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.
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