Using the New England Biolabs IMPACT system, we have expressed our protein of interest with a C-terminal fusion of a chitin binding domain (vector is pTXB1 from the NEB IMPACT system). The protein expresses well in E. coli and, judged by SDS-PAGE, has the right molecular weight (66 kDa + 27 kDa chitin binding domain tag). However, when we use the recommended protocol, our fusion construct does not bind the chitin resin, either when loaded onto a column via gravity flow or in batch when mixed with the resin and allowed to gently shake for at least an hour. Anybody observe this problem before? What could cause the chitin binding domain to fail to stick to the chitin resin?
Try to use low ionic strength buffer during adsorption or affinity binding of chitin to the column matrix. Also check pH either to low or high pH prevent the binding, and aggregated protein may be an issue!
Hello Matthew,
The first thing to be done is to see where the problem resides; why isn’t your protein binding to the chitin resin.
First, did you make sure that your protein is in fact not binding or are you assuming that it didn’t? What I mean by this is : Did you make a 2D gel of the protein solution after incubation with the resin to verify that it is indeed still in the solution ? (or maybe an antibody dot blot?) If not, you should do that. If the protein isn’t still in the solution it means that it has been binded and that your problem is in the cleavage (the last step, releasing the protein) and not the binding.
If you did indeed verify that the protein isn’t being fixed on the chitin resin, then this could be due to one of the following:
1- Your intein domain insertion wasn’t in respect to the reading frame, that could result into a protein of the right size but lacking the functional intein protein sequence (less likely because there is low chance of not having a stop codon in a 27kDa protein)
Troubleshooting: verify the integrity of the construct by bioinformatics
2- The protein sample hasn’t been washed enough, leaving detergent traces that inhibit the interaction between your intein tag and the chitin resin. (only possible if lysis method involves the use of SDS or other ionic detergents) Troubleshooting: add an additional washing step to your protein extract.
3- The protein sample hasn’t been washed enough, leaving traces of reducing agent (from protein extraction protocol, if applicable) that inhibit the interaction between your intein tag and the chitin resin. (only possible if lysis method involves the use of reducing agents like Bme/DTT/DTE) Troubleshooting: add an additional washing step to your protein extract. You could also add 1% volume of a 30% Hydrogen peroxide to your washing buffer.
4- High ionic content of the column binding solution (the solution you dissolve your protein in prior to incubation with resin) preventing the interaction between the intein tag and chitin resin. Troubleshooting: Reduce the concentration of monovalent cations in the solution.
5- High stringency of the wash buffer, leading to the dissociation of the intein from the chitin resin. Troubleshooting: Lower the concentration of monovalent cations in the solution.
6- Washing buffer pH isn’t adequate to the zwitterionic charge needed for the intein to bind the chitin resin. Troubleshooting: verify pH of your solution and check the KIT’s datasheet.
7- Protein concentration too high, causing protein agglomeration and the masking of intein tag. Troubleshooting: try diluting your protein by factor of 10.
8- The intein tag is encapsulated inside domains of your protein making it inaccessible to chitin beads. Troubleshooting: insert 25 glycine moieties between the coding sequence of your protein and the coding sequence of the intein.
I hope this helps,
Best of luck
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