Paul.xu at 19:51, 21 March 2012

2012-03-21T19:51:49Z

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IMPACT Vectors and Applications

{| cellpadding="1"
|- valign="top" bgcolor="#eaeaea" align="center"
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Vectors'''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Site of Target Protein Fusion'''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Intein Tag (kDa)'''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Recommended Cloning Sites'''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Preferred Residues at Cleavage Sitea '''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Method of Cleavageb,c '''
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Applications'''
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6707.asp pTXB1] [http://www.neb.com/nebecomm/products/productN6708.asp 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
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6709.asp pTYB21] [http://www.neb.com/nebecomm/products/productN6710.asp 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
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6701.asp pTYB1] [http://www.neb.com/nebecomm/products/productN6702.asp pTYB2] [http://www.neb.com/nebecomm/products/productN6703.asp pTYB3] [http://www.neb.com/nebecomm/products/productN6704.asp pTYB4]

| C-terminus
| Sce VMA1 intein (56)
| nowrap="nowrap" | 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)
| valign="middle" rowspan="3" |
DTT (or MESNA) pH 8.0-8.5, 4°C

| Purification and ligation
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6951.asp pTWIN1]

| C-terminus (Intein 2)
| Mxe GyrA intein (28)
| nowrap="nowrap" | NdeI-SapI (or SpeI)
| Y, F, Q, N, T, K, A, H, M, LEM (Unfavorable residues- S, P, D, G)
| rowspan="2" | Purification; C-terminal thioester for ligation and modification
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6952.asp pTWIN2]

| C-terminus (Intein 2)
| Mth RIR1 intein (22)
| nowrap="nowrap" | NdeI-SapI (or SpeI)
| G, A, LEG (Unfavorable residues- P, E, D)
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6951.asp pTWIN1]d

| N-terminus (Intein 1)
| Ssp DnaB mini-intein (27)
| nowrap="nowrap" | 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
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6951.asp pTWIN1]d

| 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)
| rowspan="2" | Step 1: pH 6.0-7.0, 25°C Step 2: DTT (or MESNA) pH 8.0-8.5, 4°C
| rowspan="2" | Purification; Defined N-terminus (e.g. Cys); Ligation and Cyclizatione
|- valign="top"
| valign="top" |
[http://www.neb.com/nebecomm/products/productN6952.asp pTWIN2]d

| 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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NEB IMPACT - Revision history

Paul.xu at 19:51, 21 March 2012