I am working with cellulases, I have expressed my cellulase enzyme in pET28a expression vector. The bacterial lysate has activity against cellulose substrate but after purification of protein with his-tag using GE 1ml His-trap column, my enzyme does not have activity. I elute my protein with 250mM imidazole, but I have dialysed my sample and still i could not find activity. I need some valuable suggestion.
Dear Muhammad,
there are a few things you need to check.
1. does your protein actually bind to the Ni-NTA column? Test the activity of the flow-throuh of the column.
2. is the protein eluted from the column? Measure the protein concentration of you eluate, or put in on an SDS-page gel. It could be that your protein remains bound to the column, or it can be that it has lost activity.
Dear Yves!
The flowthrough has activity and I have confirmed my protein elution with SDS_PAGE with the exact size. I dont know how it lose activity. Do you have some expertise?
Dear Muhammad,
It may not be 'losing' activity - instead, it is possible it may not be active in the first place, due to misfolding, and there is something else in your lysate that is showing cellulose activity. One way to check for this would be to grow another culture without inducing expression of your protein of interest, and perform the same assay with that uninduced cell lysate to see if it shows activity. You should also run the uninduced lysate on SDS PAGE to check for any leaky expression, just in case. It would be a very useful and necessary control in any case.
Lack of activity could suggest misfolding, or maybe degredation of the protein during the purification process. Have you checked to see whether any of this is happening? It may be difficult to get access, but a 1H NMR spectrum of the purified protein of interest could tell you if it's folded or not (although not if folded correctly, that's what an assay is for). Degredation could be identified through SDS PAGE or mass spec. Also, gel filtration may be useful to see if there is any oligomerisation occurring that may inhibit activity. SDS PAGE does not necessarily reflect oligomeric state. If any of these things are identified, you might consider an alternative expression vector.
Dear Muhammad,
An additional comment... it may also be the dialysis process that is the problem. Check to make sure you are using the appropriate MWCO dialysis tubing, or alternative buffer-exchange methods (desalting column, dialysis cassettes, spin-concentrators)
Dear Pryank,
Thanks alot for suggestion, actually i have transformed an empty vector without my gene of interest and performed a control activity test. And the lysate was not active, but if you suggest and this could be helpful to use an un-induced sample, i would do it this way. Other this is that I have not dialysed my sample, since the purified sample was not active. Do you think it could be the imidazole interference?
Do you think its possible that you are dialyzing away a metal cofactor?
I checked the purified protein as it is, and after dialysis too. In both cases, i had no activity.
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