There are many things to consider:

1. As purified proteins become more purified and dilute they may become less stable toward oxidation or denaturation. Consider including stabilization agents such as salts (e.g. NaCl), and/or polyols (glycerol, PEGs)

2. You may be purifying away a co-factor (e.g. coenzyme or metal ion). Replace cofactors for activity analysis.

3. The protein may be proteolyzed. Include protease inhibitors or purify proteases away from the protein as soon as possible. Check for protelolysis by SDS-PAGE.

4. If reduced Cys residues are important for activity or structure, you may have to include reducing agents to stabilize reduced enzyme, e.g. BME, DTT, TCEP. The presence of metal ions will accelerate thiol oxidation, and can be suppressed with chelators like EDTA (1-10 uM).

Urea is a chaotropic agent and may (irreversibly) denature your protein. Use care here until you know more about the reversible folding/unfolding properties of your protein.

Does the crude extract retain activity with time ? If so you may need a surfactant or carrier protein in your purified material to prevent aggregation and/or adsorption to tubes etc.What is the reaction the enzyme catalyses ? The loss of activity could be related to chemistry of the active site, particualrly true of oxidative mechanisms.

Yes, there is definitely the involvement of active site, during tagging. You can use some alternative protein for tagging which involves binding of different groups.

what was the side of your tagging? C terminal or N-terminal? Sometimes one of them causes the decreasing enzyme activity.

if the cell free extract shows activity maybe your protein needs for its activity some other molecule, ion or anything else in cell free extract

The active site is definitely altered or hindered by the tagging process. The reaction catalyzed may give some idea about the active site. However, your primary problem is loss of activity on purification. The specific activity should actually increase on purification. So reassess the purification protocol followed and modify accordingly. Determination of sequential activity during stages of purification may suggest some solution for the problem.

Proteins that are of limited stability are often stabilized by adding solutes such as sucrose,glycerol, or trimethyl amine N-oxide. The latter is particularly useful to offset the denaturing effect of urea.

It is not clear how much urea you are using after cleaving off the MBPS tag. If you use 4 M or about, most likely you have denatured the enzyme you want. If that's true, it will take a series of experiments using different buffers and dialysis to see if it is possible to refolding the protein to restore activity. Also, keep track at all steps of the amount of protein and scan the absorb acne peak to confirm that you are truly following protein, which should have a peak at 280 nm, plus a shoulder at 295 nam if you have Trp residues in your sequence. Ben Dunn

There are many things to consider:

1. As purified proteins become more purified and dilute they may become less stable toward oxidation or denaturation. Consider including stabilization agents such as salts (e.g. NaCl), and/or polyols (glycerol, PEGs)

2. You may be purifying away a co-factor (e.g. coenzyme or metal ion). Replace cofactors for activity analysis.

3. The protein may be proteolyzed. Include protease inhibitors or purify proteases away from the protein as soon as possible. Check for protelolysis by SDS-PAGE.

4. If reduced Cys residues are important for activity or structure, you may have to include reducing agents to stabilize reduced enzyme, e.g. BME, DTT, TCEP. The presence of metal ions will accelerate thiol oxidation, and can be suppressed with chelators like EDTA (1-10 uM).

Urea is a chaotropic agent and may (irreversibly) denature your protein. Use care here until you know more about the reversible folding/unfolding properties of your protein.

Graham, It does as long as it is maintained at 4 C. Also, while expected for freeze thaws, activity lose of concentrated cfe stock drops 50% each time.

I may try a surfactant then.

The enzyme is thought to be bifunctional, it traditional works as a PRA isomerase but has been hinted to have gtp cyclohydrolase activity.

Ketna, I will explore my options but other tags do not help contribute to the proteins solubility.

Pinar, it is n terminal tagging, I have cleaved it and activity slightly increased, although SDS PAGE shows the active cfe fractions have little to no cleaved protein in them, well sds page quality observable. I am exploring options of other ions, etc now as well.

Ranadeep and Irwin, I will look more closely at my protocol, I will admit, I only used stabilizing agents in the growth step to influence solubility and cell stress. I will attempt to implement changes including perhaps more prolonged use of sorbitol and glycerol.

Ben, I have repeated checked my protein quality and concentration utilizing bradford, uv-vis, as well as sds page. I do use 300 mM Urea. .

Roger, I have been using your 3rd and 4th suggestions, and in the middle of exchanging cofactors. I have yet to increase my salt though, I am going to consider that suggestion as well.

Thanks for all the advice. I appreciate the help, this has already been 100 times more helpful than my lab meetings haha.

Another question, I have also noticed my protein become insoluble with the addition of DTT, hence another reason I use 300 mM Urea. I know BME is a weaker reducer would that also be less harsh to the protein?

I take it that as a chlamydial protein, it's normally cytoplasmic? If so, it's odd that DTT affects the solubility *unless* the DTT is chelating a metal ion required for stability (while there are cytoplasmic proteins with disulfides, I think they're pretty rare). BME may show less chelating activity. You could also try reduced glutathione.

Using a longer linker (multiples of GGGGS are used for some purposes) or a smaller fusion protein such as ubiquitin or SUMO or the B1 domain from protein G (GB1) might help.

I assume you've tried various nucleotides and cyclic nucleotides (which might require Mg++ for activity) in the purified protein assay?

few possibilities are there

1- your purification method including temperature and PH. check if they are same at which your protein has activity or not

2- your purified protein is not the one your want. i mean wrong protein your purified

3- the time of purification also matter may be your protein is not long lasting check this our.

Good luck

Dear James,

What I could get from your statement, is to simulate the environment of the extracted enzyme-protein sample to the assay mixture. If there is any slight deviation, it would certainly be altering or diminishing the activity status. if you are satisfied with this clue, then it is OK, otherwise further queries are welcome.

@ Manghaiko Mayele: Thanks. I would be grateful if you could please give the reference of this very important paper on the effect of urea on hydrophobic interactions (and by inference, on protein stability).

Looking forward to hearing from you soon,

With kind regards,

Sanjay

@ Manghaiko Mayele: Thanks for your prompt response. I will go through the same followed by discussion with you in this relevance very soon.

@ Manghaiko Mayele: Thank you as well, I will be read this.

I will update more in the next day or two. Regrowing and purifying my protein this weekend in different stabilizing solutions and implementing suggestions given is taking me a bit of time so I have no feedback from it yet.

@Muhammad buzdar: I am fairyly certain it is my protein, I check the plasmid prior to expression, checked the protein pre and post factor Xa cleavage of the fusion and the kDa appear to match up. Also, the pH has been maintained and I use positive controls to make sure my reagents are in working order.

@Wes Mcdermott: It is cytosolic, It may very well be chelating the metal as I tried a different metal, in which the addition of either as the last component of a reaction assay caused precipitation (in this case, maybe it is not the protein, I have not run the gel to confirm). I may try multiple nucleotides, although specifically it should only be targeting GTP.

Update. . .sort of: Well, for anyone that was still curious about this issue. I tried, high salt, glycerol, different metals, reducing agents, sorbitol/betaine (growth conditions), PEG for purification and addition of cofactors, non-reducing and reducing conditions, and running assays with and without the MBP tag.

30% Glycerol appears to retain the activity, I may try a second glycerol purification but with less. I find it has a much smaller degree of activity compared to the CFE.

I have suspected that maybe it is unfolding during the purification process and I thought maybe chaperones were necessary for proper refolding, but I am not sure how to verify this thought.

On a plus side, the majority of the conditions do not precipitate the protein, but it may be soluble in a kinetic trap. I am at a loss for words at this point.

If it’s this touchy and your main target of research, maybe it’s time to do a 180 and go for a grand supply with minimal purification compromise: can you make a monoclonal antibody? Affinity beads would be ideal to pull your stuff from a lysate, although you wouldn’t be able to do it with chaotropic agents in the mix.

It may seem like a lot of work, but not nearly as much as trying to identify cofactors, conformation and conditions that are stymying you so far.

Dear Kristine,

I am pretty sure that your suggestion would be helping out James and others facing this critical problem. In my opinion, this is very common feature concerning with membrane bound proteins. In most of the cases, affinity chromatography is observed to be only and very effective technique to be taken into consideration. Further, a compromise should be made in the in vivo and in vitro environment. For further solution, the following paper should be gone through:

Sanjay Mishra and Yasushi Kamisaka (2001). Purification and characterization of thiol reagent- sensitive glycerol-3-phosphate acyltransferase from the membrane fraction of an oleaginous fungus. Biochemical Journal 355: 315-322