G'day Tomas, it sounds like you are dealing with a difficult project here - trying to follow protein enrichment from crude extracts with no bioassay is impossible. Given that the protein is novel there are no other tests (ie ELISA or immunoassay) that you can do to track your protein. I think however, you can benefit from the following approaches. Also you probably have no sequence information either?!

Your activity disappears quickly so why not try running your crude extract on Native Page or a calibrated size exclusion column and immediately test the sectioned gel or SEC fractions for activity. That should give you a "ball park" idea of the size of your protein. Amass much SEC fractionated material with activity and then aliquot and -80*C freeze it for material to do small-scale optimisations on...

These first steps are crucial for your proteins structural integrity and hence activity. Try studying your protein more carefully one-step at a time with some controlled multiple variables - if you have the time. For example, do your extractions +/- EDTA, try spiking -EDTA extracted samples with different divalent cations (Zn, Cu, Fe, Co etc) and then test all samples (ie +/- EDTA plus -EDTA transition metal spiked samples) for desired activity. Do the same for co-factors that are known to be involved in mechanisms of other enzymes catalysing the same reaction(s) as the one your activity assay is based upon (ie NAD+/NADH etc for oxidoreductases or whatever....).

Conversely, some proteins can be poisoned by metals and other molecules, try dialysing your crude extracts to remove these LMW compounds, use EDTA/EGTA if you determine that divalent cations are poisonous and employ generous amounts of protease inihibitor cocktails also.

Do these tests for pH, ionic strength, stabilisers (glyderol, TMAO, arginine or whatever) and soon you will build up a picture of how your protein likes to be treated.... following the activity stability over days at different temperatures should therefore tell you if you have devised a way to at least keep your protein happy long enough to follow it through a suitable purification scheme

Once you have stable activity, then further purification techniques with non-purified controls (to allow for time dependent losses of activity to be factored in) can be trialled on a small scale. If you lose activity then you can ask some really cool questions: is the technique: pH, ionic strength, lack of elution, lack of adsorption telling me something about my protein and what it doesn't like? You can then try alternatives modes of chromatography, dialysis etc to purify your protein whilst simultaneously maintaining activity at similar or higher specific activity.

Don't forget that a sudden loss of activity can be a means of insight, ie size exclusion under reducing conditions can lead to covalently associated subunits being dissociated and enzyme activity being lost... Don't give up in these times because there is vital information lurking under the surface... each technique has its own physico-chemical basis and therefore in fact reports on the physico-chenmical nature of your analyte and its activities.

Good luck and keep me posted because this sounds really cool.

The problem is that it is brand new enzyme, so there are no papers about it, no database entries, no gene info etc. That's what I should make :-D

So, I'm purifying endogenous protein from maize and the activity was like fkat/ml in crude extract, so really not much after O/N reaction.

I guess I cannot extrapolate the optimal conditions from activity measurement for extract, right?

I was told to use reducing conditions (mercaptoethanol) and glycerol, so I will try and see...

Freeze small aliquots at -80 is a great suggestion. Try to minimize the freeze/thaw cycles. Anything that makes your buffer more polar may help to stabilize your protein. Good luck!

Too few bonds between the secondary structures such that they are broken by minimal heat and/or there could be a specific ion required for stability of the enzyme. Hydrogen bonds and salt bridges contribute the most to thermostability. Design an assay to compare the activity of your enzyme across different TYPES of salts at a specific temperature and incubate your protein over fixed periods. After you get the ion that ensures high activity, test for varying concentrations then move to temperature. Check the publication: Factors affecting thermostability by Kumar et al., 2000.

Hi,

thanks all for the responses.

The problem is that I do not have purified enzyme. I'm trying to purify it, but I've lost the activity meanwhile.

The thing is that I do not need to store the protein. If it was about storage I wouldn't ask, but I need to work with the protein.

My procedure was as follows:

extraction

precipitation

Octyl Sep

concentration and desalting on centrifugal filter devices

High Q

some concentration

metal chelating chromatography

this took me two to three weeks and after last step I was not able to detect any activity.

What do you see on PAGE? Is the loss of activity due to protease activity in your sample?

Usually proteolysis happens in not pure protein solution at 4oC

If YES, than try to add inhibitors, that should help. The other way is to add "buffering" protein to the solution, as BSA, for storing at 4oC.

Your purification scheme is not very long, why does it takes weeks to complete it?

I used to work with an oligomeric enzyme that was unstable on ice, especially in the presence of salt, but was quite stable at room temp. The entire purification was done at room temp. To store the protein, we precipitated it with ammonium sulfate and stored the precipitate at 4C. To use the protein, we spun down the precipitate at 4C then resuspended it in buffer at room temp. The ammonium sulfate was removed using gel filtration spin columns.

G'day Tomas, it sounds like you are dealing with a difficult project here - trying to follow protein enrichment from crude extracts with no bioassay is impossible. Given that the protein is novel there are no other tests (ie ELISA or immunoassay) that you can do to track your protein. I think however, you can benefit from the following approaches. Also you probably have no sequence information either?!

Your activity disappears quickly so why not try running your crude extract on Native Page or a calibrated size exclusion column and immediately test the sectioned gel or SEC fractions for activity. That should give you a "ball park" idea of the size of your protein. Amass much SEC fractionated material with activity and then aliquot and -80*C freeze it for material to do small-scale optimisations on...

These first steps are crucial for your proteins structural integrity and hence activity. Try studying your protein more carefully one-step at a time with some controlled multiple variables - if you have the time. For example, do your extractions +/- EDTA, try spiking -EDTA extracted samples with different divalent cations (Zn, Cu, Fe, Co etc) and then test all samples (ie +/- EDTA plus -EDTA transition metal spiked samples) for desired activity. Do the same for co-factors that are known to be involved in mechanisms of other enzymes catalysing the same reaction(s) as the one your activity assay is based upon (ie NAD+/NADH etc for oxidoreductases or whatever....).

Conversely, some proteins can be poisoned by metals and other molecules, try dialysing your crude extracts to remove these LMW compounds, use EDTA/EGTA if you determine that divalent cations are poisonous and employ generous amounts of protease inihibitor cocktails also.

Do these tests for pH, ionic strength, stabilisers (glyderol, TMAO, arginine or whatever) and soon you will build up a picture of how your protein likes to be treated.... following the activity stability over days at different temperatures should therefore tell you if you have devised a way to at least keep your protein happy long enough to follow it through a suitable purification scheme

Once you have stable activity, then further purification techniques with non-purified controls (to allow for time dependent losses of activity to be factored in) can be trialled on a small scale. If you lose activity then you can ask some really cool questions: is the technique: pH, ionic strength, lack of elution, lack of adsorption telling me something about my protein and what it doesn't like? You can then try alternatives modes of chromatography, dialysis etc to purify your protein whilst simultaneously maintaining activity at similar or higher specific activity.

Don't forget that a sudden loss of activity can be a means of insight, ie size exclusion under reducing conditions can lead to covalently associated subunits being dissociated and enzyme activity being lost... Don't give up in these times because there is vital information lurking under the surface... each technique has its own physico-chemical basis and therefore in fact reports on the physico-chenmical nature of your analyte and its activities.

Good luck and keep me posted because this sounds really cool.

Have you tried adding Some stabiizers to your protein? also you could use a protease inhibitor that might help!

Try all the above said things once you are sure, that u r getting protein after your purification steps. Confirm it by a inmunoblot or mass spec, which ll ensure you r in a right tract!

i agree with Mr. Sunil. your protein but be undergoing degradation due to protease. so i will suggest that to purify your protein at a strech without any delya after the cell lysis. and do not forget to add protease inhibitor at every step if neccessary. And try doing each step in ice cold condition. hope this will work out..... ALL THE BEST.....