Go to New England Biolab's web site to download the instruction manual. There is also an excellent FAQ for the system which provides lots of information.
IMPACT, Intein-Mediated Purification with an Affinity Chitin-binding Tag, is a novel protein purification system that allows recombinant proteins to be purified without affinity tag in a single chromatographic step. This method was developed at New England Biolabs (NEB) from studies of the mechanism of protein splicing (see references). It distinguishes itself from all other purification systems by its ability to purify a recombinant protein with its native sequence by a single affinity column, without the use of a protease. The IMPACT system
utilizes the inducible self-cleavage activity of an engineered protein splicing element (intein) to separate the target protein from the affinty tag. The target protein is fused to a tag consisting of the intein and the chitin binding domain which allows affinity purification of the fusion precursor on the chitin column. The intein is induced by thiol reagent or pH and temperature shift to undergo specific cleavage which releases the target protein from the chitin-bound tag resulting in a single column purification of the target protein. The IMPACT system includes a series of E.coli expression vectors which utilize engineered inteins of 134-454 amino acid residues. These vectors are designed for protein expression and purification in E.coli as well as protein manipulations such as protein labeling, ligation and cyclization.
(Taken from the NEB website).
The Impact system has proven useful in our lab for purifying proteins that have a C-terminal hydrophobic sequence, since fusing these proteins to a C-terminal affinity tag masks the hydrophobic sequence, increasing solubility of the fusion.
Vectors available in the lab
For further details on these vectors, and other vectors available from NEB, see the FAQ (http://www.neb.com/neb/frame_tech.html)
pCYB1-4 (plasmid #1066, 1067, 1068, 1069)
These vectors aren't sold by NEB anymore, but are available by special request. Expression of the protein-intein-CBD fusion gene is under the control of the tac promoter. Except for the promoter difference, these vectors are identical to the corresponding TYB1-4 vectors described at the NEB site. The difference between CYB1, 2, 3, and 4 is in the multi-cloning region.
Cloning using the Sap1 site places the target protein directly adjacent to the intein, with no additional amino acids in between. Cloning using the Sma1 site produces a target protein with a glycine added to the C-terminal end.
pTYB3 (plasmid #1179)
Expression of the fusion gene is under the control of the T7 promoter. It is otherwise identical to pCYB3
pTXB1 (#1209), pTXB3 (#1210)
The TXB vectors use a different, smaller intein (the Mxe GyrA intein-198 aa ~20-25 kD). In some cases smaller inteins result in higher expression, and there may be less in vivo cleavage with these vectors. There are some differences from the Sce VMA intein (CYB, TYB intein) in terms of favourable residues at the -1 position (last amino acid of your protein). Gly or Ala in this position slows down the cleavage reaction, and Ser or Pro blocks it completely.
Also not commercially available from NEB. They use a different mini-intein (Ssp dnaB mini-intein, 154 aa, 17 kD). NEB says that cleavage may be induced by shifting pH (pH6 to pH8-9) and temperature (4oC to 20oC) in the absence of DTT. Purification should be carried out at 4oC at pH6, while on-column cleavage should be performed at pH8, room temperature, overnight. (This hasn't been tested in our lab). Cleavage can also be carried out in the regular manner. In vivo cleavage is a problem with these vectors, induction should be carried out at low
temperature (12-15oC).
Multi-cloning region
TSB21 -Nco1-Sal1-Not1-EcoR1-Xho1-Sap1-
TSB22 -Nde1----------------------------Sap1-
Cloning
The IMPACT instruction manual describes how to design primers for cloning into the IMPACT vector, and gives examples of what the primers might look like. Be aware that the Sap1 site is lost following cloning.
****These vectors must be grown in bacteria that are r-m-. Using a strain with wild type EcoK (hsd+) will result in losing your DNA. DH5 cells work well.
The amino acid directly N-terminal to the first Intein residue (cys) can affect the cleavage reaction. Find out the last residue in your protein and check the manual for what effect it may have.
These vectors are low copy number. Large scale plasmid preps from 100mL of SB typically only give about 20-30ug of DNA. Do a larger volume or use some kind of plasmid amplification procedure. For minipreps, double the amount of culture you usually miniprep.
Sequencing
We have some primers for sequencing clones that map positive.
T7 Universal primer - forward sequencing of TYB and TXB inserts. Anneals to the T7 promoter region
Tac primer (#672) - forward sequencing of CYB inserts. Anneals to the Tac promoter region
Intein reverse primer (#625) - reverse sequencing of CYB and TYB inserts. Anneals ~60 base pairs downstream of the cleavage site.
TXB reverse primer (#573) - reverse sequencing of TXB inserts. Anneals ~100 base pairs downstream of cleavage site.
Protein expression and purification
Overexpression is typically carried out by growing the cells to OD600 of 0.6, then inducing for 3 hours with 1mM IPTG. If using the fermenter, grow the cells to OD of ~0.7-1 before induction.
Expression from the CYB vectors can be carried out in DH5 cells. For the TYB and TXB vectors use BL21(DE3) cells.
Following induction the cells can be stored as frozen pellets. For cell lysis, pellets should be resuspended in Column buffer, and can be lysed using mechanical methods such as the French Press. Don't use lysozyme, it will degrade the chitin column.
If you are using the low-tech column approach (gravity-run) be aware that these columns run SLOWLY. Make the column bed as wide and short as possible, and start early in the morning. It often works better to run, for example, 2 2-ml columns in parallel, rather than 1 4-ml column.
After loading and washing the chitin column with Column buffer, wash with 5 bed volumes Cleavage buffer if the NaCl concentration differs from the Column buffer. Then wash the column with at least 2 volumes of Cleavage buffer + cleaving agent (e.g. DTT). Don't let this wash go longer than 15 to 20 minutes, or your protein may start coming off the column. Following cleavage, elute the protein with Cleavage buffer (without cleaving agent)
Due to the chemistry of the cleavage reaction, the cleaving agent (DTT, cysteine, -mercap, hydroxylamine) will remain on the C-terminal end of the protein. Be careful that its presence will not interfere with future uses of your protein. I.e. if you are going to label your protein with a cysteine-specific label, don't use DTT or cysteine, it will add a free sulfhydril group to the end of the protein. Use hydroxylamine instead. Cleavage conditions used successfully in our lab are:
30 mM DTT overnight at 4ºC
100 mM hydroxylamine overnight at 4ºC. Be sure to pH the solution back up to ~pH6-7 with NaOH after dissolving the hydroxylamine or it won't work.
The different inteins and different cleavage conditions may have different cleavage kinetics. If it won't cleave overnight at 4ºC, try two nights, or at room temperature, before giving up.
Regenerating the column
Wash the column AT ROOM TEMPERATURE with at least 10 bed volumes of stripping buffer (recipe given below). Don't do this at 4ºC and don't try to store the buffer long-term, it will precipitate in both cases. Then wash the column with lots of dH2O (at least 10 volumes) and then store it in Column buffer. Add 0.02% azide for long term storage.
Buffers
These are standard recipes for the buffers used for cell lysis and column purification. The buffers can be modified by the addition of glycerol (up to 20% causes no problems) or various detergents (check the FAQ, up to 0.2% CHAPS does not affect purification). The NaCl concentration can be increased up to 2M according to NEB (we've done 1M) for more stringent washing.
