Editing NEB IMPACT

IMPACT Vectors and Applications 

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| nowrap="nowrap" bgcolor="#c0b0b0" | '''Vectors''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Site of Target<br> Protein Fusion''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Intein Tag<br> (kDa)''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Recommended<br> Cloning Sites''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Preferred Residues at Cleavage Site<sup>a</sup> ''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Method of Cleavage<sup>b,c</sup> ''' 
| nowrap="nowrap" bgcolor="#c0b0b0" | '''Applications'''
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[http://www.neb.com/nebecomm/products/productN6707.asp pTXB1]<br>[http://www.neb.com/nebecomm/products/productN6708.asp pTXB3] 

| C-terminus 
| Mxe GyrA<br> intein (28) 
| NdeI-SapI (or SpeI) 
| Y, F, Q, N, T, K, A, H, M, LEM<br> (Unfavorable residues- S, P, D, G) 
| DTT (or MESNA)<br> pH 8.0-8.5, 4°C 
| Purification; C-terminal<br> thioester for ligation and modification
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[http://www.neb.com/nebecomm/products/productN6709.asp pTYB21]<br>[http://www.neb.com/nebecomm/products/productN6710.asp pTYB22] 

| N-terminus 
| Sce VMA1<br> intein (56) 
| SapI-PstI<br> BsmI (or NdeI)-PstI 
| A, Q, M, G, L, N, W, F, Y<br> (Unfavorable residues- P, S, C, T, R) 
| DTT<sup>c</sup><br> pH 8.0-8.5, 25°C 
| Purification
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[http://www.neb.com/nebecomm/products/productN6701.asp pTYB1]<br>[http://www.neb.com/nebecomm/products/productN6702.asp pTYB2]<br>[http://www.neb.com/nebecomm/products/productN6703.asp pTYB3]<br>[http://www.neb.com/nebecomm/products/productN6704.asp pTYB4] 

| C-terminus 
| Sce VMA1<br> intein (56) 
| nowrap="nowrap" | NdeI-SapI<br> NdeI-SmaI (or XhoI)<br> NcoI-SapI<br> NcoI-SmaI (or XhoI) 
| G, A, M, F, Q, W, Y, K, LEG<br> (Unfavorable residues- P, C, N, D, R) 
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DTT (or MESNA)<br> pH 8.0-8.5, 4°C 

| Purification and ligation
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[http://www.neb.com/nebecomm/products/productN6951.asp pTWIN1] 

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

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

| N-terminus<br> (Intein 1) 
| Ssp DnaB<br> mini-intein<br> (27) 
| nowrap="nowrap" | SapI-SapI<br> SapI-PstI (or BamHI)<br> BsrGI-PstI (or BamHI) 
| C, S, A, G, M, T, CRAM<br> (Unfavorable residue- P) 
| pH 6.0-7.0,<br> 25°C 
| Purification; Defined<br> N-terminus (e.g. Cys);<br> Ligation
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[http://www.neb.com/nebecomm/products/productN6951.asp pTWIN1]<sup>d</sup> 

| N-terminus &amp;<br> (Intein 1)<br><br> C-terminus<br> (Intein 2) 
| Ssp DnaB<br> mini-intein (27)<br><br> Mxe GyrA<br> intein (28) 
| SapI-SapI 
| C, S, A, G, M, T, CRAM<br> (Unfavorable residue- P)<br><br> Y, F, Q, N, T, K, A, H, M, LEM<br> (Unfavorable residues- S, P, D, G) 
| rowspan="2" | <br><br> Step 1:<br> pH 6.0-7.0, 25°C<br><br> Step 2:<br> DTT (or MESNA)<br> pH 8.0-8.5, 4°C 
| rowspan="2" | Purification; Defined<br> N-terminus (e.g. Cys);<br> Ligation and Cyclization<sup>e</sup>
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[http://www.neb.com/nebecomm/products/productN6952.asp pTWIN2]<sup>d</sup> 

| N-terminus &amp;<br> (Intein 1)<br><br> C-terminus<br> (Intein 2) 
| Ssp DnaB<br> mini-intein (27)<br><br> Mth RIR1<br> intein (22) 
| SapI-SapI 
| C, S, A, G, M, T, CRAM<br> (Unfavorable residue- P)<br><br> G, A, LEG<br> (Unfavorable residues- P, E, D)
|}

<sup>a</sup> Actual cleavage efficiency is dependent on the adjacent residues as well as the folding of the fusion protein. <br><sup>b</sup> 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. <br><sup>c</sup> Cysteine can be used in the place of DTT.<br><sup>d</sup> pTWIN vectors allow for induction of intein cleavage by a pH/temperature shift.<br><sup>e</sup> 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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