Hi there,

I don't think cell pellet freezing has anything to do with protein inactivation. If you have a functional assay you may run it at different stages of the extraction/purification protocol in order to identify where activity is lost. In my experience we systematically freeze cell pellets as it improves the lysis efficiency and I have never observed protein inativation due to this process.

It is possible to lose protein activity upon freezing a cell pellet, although very unlikely. You should check activity before and after freezing to ensure that your protein is expressed at high level AND functional.

Dear sir , my experience in this field freezing does not affect on  the protein activity

Thank you guys for your input. To Dominique, I have recently checked the functionality of my protein after ni-nta purification - and do see the intended reaction; however, the speed it generates the product does not seem to be as fast or as efficient as the literature reports.

Will continue troubleshooting. Carlo's answer was what I needed, so I will try to express and purify without freezing my pellet and see if there the functionality of my protein is preserved.

Hi,

Beth from Practichem here.  I agree with Dominique and the other responders, and I have used repeated freeze-thaw cycles to facilitate lysis when working with small pellets/volumes or when the cell-cracker is broken.  I am glad you took his advice and checked the activity.  I wanted to point out that the reduction in activity may be due to the presence of the poly-histidine tag itself.  It is not uncommon for a tag to affect local structure such that activity or substrate binding is reduced.  Additionally, if your assay relies on metals, then the concentration may need to be raised to compensate for the metal ions that will bind the tag.  I highly recommend using a protease such as TEV to remove the histidine tag.  Then you can compare pre and post-cleavage activity.  It's better to do this kind of analysis early, so you can be sure any later results are not artifacts due to the tag.  I have seen papers retracted because the effect of a small protein tag significantly altered protein activity.   

Please feel free to send a message if you have any questions, or if you found my advice particularly helpful.  I would certainly be interested to know if the tag affected the activity or not.

Good Luck!

Beth 

I agree with Beth. The His tag could definitely be causing problems with activity. Try a pre- and post-tag cleavage activity study.

Also, are you absolutely sure you have your target protein and that it's as pure/homogeneous as you think? (Have you gotten confirmation with a western blot and/or peptide fingerprinting, etc.?) You could have a contaminant coming through at the same band/size, or misfolding taking place, etc.

Hi Elizabeth and Jesse.

I recently tried re-expressing and purifying in one go (without freezing the pellet), activity is still quite poor.

To Elizabeth; I am considering designing a non-his tag version of the protein to make the comparison.

To Jesse; there are very few contaminants after my two steps purification. I did NiNTA affinity, then I performed size exclusion. Misfolding is a possibility - which may point me in the direction of not having a his tag. Western blot is something I would consider also to check if it's really my protein that I am purifying. So far base on SDS-PAGE and elution profile from size exclusion - I believe that what I am looking at is my protein of interest.

Will keep you posted whenever I have functionality result on +/- his tag. Thank you again for your input.

Derrick,

If you don't have the time/resources for a western blot, you could also try using a His-tag stain. We use one (see link below) that has worked really well for us and helps eliminate the guess work a little bit. It's also easier/faster than a western blot.

https://www.thermofisher.com/order/catalog/product/LC6030

Have you tried cleaving the His-tag and re-trying the activity assay yet?

I have seen purified proteins that become inactive after freeze thaw. Usually there is some aggregation or precipitation that can be visualized on an SEC column. To avoid this I suggest flash freezing smaller aliquots in liquid nitrogen and do not thaw on ice, the slower thawing can disrupt your protein by artificially high concentration as the protein will thaw out faster than the ice.   

To Jesse Jones and Elizabeth A Mcsweeney

I finally found out what caused the inactivity. Basically there was some sort of metallo proteases and the protein was fully functional when it was eluted directly in a tube containing few uL of concentrated EDTA and subsequent functional assay shows that the protein was highly reactive (comparable to published values). Apology for the very tardive reply - as in the end I never get to use the protein in further studies.