I've clone a gene of Beta glucosidase enzyme in E.coli. Most of my protein is going in inclusion bodies. So I tried the urea treatment for the solubilization of inclusion bodies and afterwards dialysis was done to remove urea. But after dialysis the protein is not giving any activity using p-nitrophenyl glucopyranoside as substrate . However, when I tried the whole cell method (50 O.D. cells were taken) colour of the test change to faint yellow.
Is that my protein giving acitivity or is it reacting with something else leading to degradation of the substrate?
Protein refolding is tricky. Even if you end up with a soluble protein, it may be aggregated and inactive. Beta-galactosidase from E. coli is a tetramer of large monomers, so it is probably particularly difficult to refold successfully. There may have been a portion of the expressed protein that was in an active, soluble form in the whole-cell extract. Considering the difficulty of refolding, it is always worth trying to get the soluble, active protein first, and save refolding as a last resort.
There are substances you can add to the refolding buffer to improve the chance of getting something useful. L-arginine at 0.5 M is a popular choice.
Try to solubilize the inclusion bodies using non-denaturing surfactants such as Sarkosyl, Triton X-100, and CHAPS. This may keep the glucosidase properly folded-active. Refolding may end as scrambled structures of glucosidase which cannot reflect the accurate activity content, thus it would be ideal to solubilize the enzyme with alternative agents which are not harmful to the native form.
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