I have recently expressed recombinant nitrilase in BL21. After purification I got a single band on SDS-PAGE of desired size. I stored the protein in 10% glycerol at -80C (elution buffer had 50mM NaH2PO4,300mM NaCl, 250mM Imidazole pH=8). I went on to do a functional nitrilase assay but it did not work at any concentrations I used.
Any advice would be appreciated.
Sometime His-tag has negative effects on the protein folding and its activity. You could try to use a His-tag on the other terminus of the protein, or to remove the His-tag after purification. More helpful can be another tag or a different purification method.
Also: Do you know if the protein is active without the His-tag, do you have this as a reference? Also the buffer might be an explanation for the inactivity.
You also should check if the single band corresponds definitly to your protein. A co-purification of E. coli proteins is very common using His-tag, for example SlyD with 21 kDa is often co-purified, as well as others, containing a few contiguous His residues.
It would be best to assay the protein before storage to make sure the problem is not stability upon keeping in glycerol at -80. The other issue is your assay and how confident you are that the assay conditions are appropriate. More info is needed.
Sometime His-tag has negative effects on the protein folding and its activity. You could try to use a His-tag on the other terminus of the protein, or to remove the His-tag after purification. More helpful can be another tag or a different purification method.
Also: Do you know if the protein is active without the His-tag, do you have this as a reference? Also the buffer might be an explanation for the inactivity.
You also should check if the single band corresponds definitly to your protein. A co-purification of E. coli proteins is very common using His-tag, for example SlyD with 21 kDa is often co-purified, as well as others, containing a few contiguous His residues.
I did look up recombinant nitrilase purification and there are articles with preparations of his tag nitrilases, but Marlen brings up a good point. Do you follow what is known in the literature for nitrilase constructs and expression? The assays they used were for adiponitrile.
If memory serves correctly I recall being told at some point that -80 storage in phosphate buffer tends to reduce enzyme stabilty. Was a paper of some description about general storage procedures and their effect on stability. Perhaps Tris is a better option? You can also try increasing the glycerol concentration. 10% feels a little low. Personally I use 40% in most cases, while 20% is common from commercial suppliers of proteins. Do you have a working sample as positive control (eg from tissue isolate or commercial source?) Perform circular dichroism on a freshly isolated sample of yours and comparing that to a working positive control can tell you about mis-folding.
Thanks Marlen...
I have a C-terminal his- tag. My protein is 37KDa and it is exactly the desired size. May be the his-tag is creating the problem, i will work on it.
Marcia......I carry the assay in phosphate buffer pH8 using benzonitrile as substrate.
I'm not completely familiar with this topic, but I stumbled across a paper with some hints how to improve your enzyme handling - http://mic.sgmjournals.org/content/151/11/3639.full
Did you consider rebuffering your enzyme for the assay or storage? As said before, some buffers may not be compatible...
Also disulfide-bridges, degradation, etc. comes to my mind when thinking of inactive enzymes...
It looks like from the paper I saw, that they used 500mM imidazlole to elute their nitrilase and then used the enzyme in the assay. But I agree that storage could be a different issue. Most people don't keep their proteins in imidazlole buffer. They do buffer exchange.
I could not find from a quick search what buffer to use. So I would try the assay before adding glycerol and freezing. If there is activity, then I would do a literature search to find buffers compatible with nitrilase and do buffer exchange, and re assay before freezing. Then I would try different storage conditions, ie without and with varying amounts of glycerol to see under what conditions your protein is stable.
If there isn't activity after the Ni NTA column, then you may want to try buffer exchange, but the issue will always be what buffer to use and this should be on line in the literature, or perhaps someone can comment.
Here is a link where you can find info. Nitrilase uses a catalytic cysteine, and DTT addition may be needed or some other reducing agent in small quantities.
Information on EC 3.5.5.1 - nitrilase - Brenda
www.brenda-enzymes.org/php/result_flat.php4?ecno=3.5.5.1
Nlase, Pseudomonas fluorescens, -, shows nitrilase and nitrile hydratase activities ... enzyme belong to branch 10 of the nitrilase superfamily, overview, 655310 ...
Your protein may be sensitive to freezing, I would make the peak column fraction 50% glycerol (gently mix with a pipette tip that has had the end cut off to make it larger to avoid shearing affects) and store the 50% stock at -20oC. Imidizole will then be 125 mM and if you dilute 1:50 into an assay it will only be 2.5 mM final. Imidizole is a buffer so it should not be a problem in terms of the protein function as long as it "does not" change the assay buffer pH. Just keep in mind the concentration of the 1X assay buffer in the reaction and the final concentration of imidazole in the same reaction (>10:1 ratio of assay buffer to imidazole should prevent any significant pH change).
Also consider whether the imidazole might be causing the loss of your magnesium cofactor.
Dialyse out imidazole and try. If it is still problem, try to have His-tag in the opposite side. This might help
In the case of a correctly synthetized protein and the purification procedure does not alter it, you would remove the His Tag. It is possible, this could alter the protein conformation and thus inactive the enzyme.
Prof. Dr. Joseph Kreit
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression