Hi everyone,
I have never worked with Inclusion Bodies so do not have a method that really works.
My protein is recombinant ß-casein that is produced both in the soluble fractions of the pellet and the insoluble ones. As for the pellet, we did resolubilization with buffer containing 8 M urea and directly load it on the HisTrap columns. The flow rate was 0.5 mL/min (the slowest on ÄKTA Start machine) but still overpressure often happened. I was pretty sure this was not the best method to purify the desired protein from the resolubilized pellet, and some kind of intermerdiate method needs to be done before purifying on the ÄKTA.
Therefore I need advice of either:
1. What I can do after resolubilization to alleviate the pressure before loading the sample to the HisTrap column? (my protein has a His Tag)
or
2. Is there any other method that can be used to purify the ß-casein? Maybe precipitation?
Column chromatography is not a must so other methods are welcome.
This is a trial and error for me so I can try any suggestions.
All suggestions and discussions are welcome.
Thank you in advance.
If there is a reasonable amount of casein in the soluble fraction, work with that. It will be much easier than working with the insoluble protein.
As for the insoluble protein, you could try refolding it by rapid dilution of the urea solution dropwise into a large volume of refolding buffer, or you can use step-wise dilution by dialysis against refolding buffer.
I spent a lot of time on this refolding a similar inclusion body (IB) and had great luck extensively washing the IBs with lower concentrations of the denaturants than were required to solubilize them. For example resuspending them in 1-2M urea and using probe sonication to re-disperse the IBs after this wash. I then pelleted the IBs and repeated. Eventually the IB's became very pure, and only then did I solubilize them and perform metal chelate chromatography. Good luck!
Edward Michelini Great idea.
I had a recent experience combining purification with on-column refolding of a His-tagged protein from inclusion bodies using an IMAC column. The inclusion bodies were dissolved in 6 M guanidine-HCl, bound to the column, washed with the same solution, refolded by linear gradient from 6 to 0 M guanidine, then eluted with imidazole. All solutions contained arginine to assist with refolding, and redox buffer for disulfide bond formation. Most of the eluted protein was aggregated, but a small portion was monomeric, had the correct disulfide bonds, and was catalytically active. The aggregated protein was removed by gel filtration chromatography.
The aspects I like about this method are (a) the amounts of solutions required are much less than the dialysis technique, (b) the procedure is automated when run on the FPLC and runs overnight, and (c) it does not require concentrating from a large volume as in the dilution method.
Hi a little update:
I tried to do on column refolding like in the literature or as suggested but didn't work it. However I did do pellet wash before loading on the column with Triton X-100, 1 M NaCl, and regular buffer. I had no pressure problem as the lysate looks clear, but the refolding on the column did not really work out as there was no protein during the elution.
Next time I will try to purify it then do the protein refolding.