NEB IMPACT

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.