I am expressing 2 enzymes in BL21, one is the WT form and the other a variant. I expressed the WT form without using any PMSF in the lysis buffer and the activity is fine. However, when I express the variant (2 aa were changed), the enzyme has very little activity. It is entirely possible that the mutations destroy the enzyme activity, but I am curious as to whether anyone has noticed decreased activity of a bacterial expressed enzyme when using protease inhibitors such as PMSF. I normally don't use protease inhibitors when working with bacteria (only use them with yeast).
Something to consider -- One of the challenges in using PMSF as a protease inhibitor is its limited solubility in aqueous solutions. Very often PMSF stock solutions are made using alcohols(1-10mM max), acetone or even ethylene glycol(~100mM max) as solvent. Since you described not using PMSF at all in the wt expression, it might be the PMSF delivery solvent that is of concern and not the PMSF itself. I have personally moved to the water soluble APMSF just to avoid this potential concern. It's definitely more expensive, but I no longer worry about potential solvent effects.
PMSF is a serine protease inhibitor and its activity is based on its reactivity on a serine with a drastically lowered pKa, which is due to the special microenvironment found in serine proteases. Of course it's possible (yet I would say quite unusual) that PMSF altered your enzyme activity. It would require to have an "activated" solute accessible serine that is crucial for your activity.
I would suggest you to assay this with the wild type enzyme to make sure.
It is possible that PMSF could react with your protein. Although it is a relatively specific setine protease inhibitor, it is also a general electrophile, and could potentially react with exposed nucleophiles in an unfolded or partially unfolded protein. It's a long shot. I've never observed this in 30 years of protein preps. In general, I find protease cocktails unnecessary in E. coli if you maintain 4 deg C and purify promptly.
You can always purify both your proteins in buffers without PMSF but includes protease inhibitor cocktail. This would probably give you a better idea about your mutant activity. Without knowledge of what is the mutation for it will be hard to say exactly.
Hi,
activity of the enzyme can change if you change also only one aa. Depends where the aa is. If you change one of the aa in the catalytic pocket the activity will drastically change. Also if the aa you changed are big they might block the access of the substrate to the catalytic pocket. It is better if you use some simulation program that shows you what happen to the enzyme when you change the 2 aa.
I use PMSF when I expressed my protein in bacteria. My protein was an aspartyl proteases and PMSF only inhibits the activity of serine proteases.
I agree with all the comments above - but you haven't mentioned what your enzyme activity is so it's difficult to say confidently whether it is possible could PSMF could be having an effect. Either way, I always use PMSF in my protein preps from e coli but never exceed 0.5mM - it is an irreversible (serine protease) inhibitor as well, perhaps if you are going above 1mM you may start to get non-specific effects. I also use protease inhibitor cocktail tablets and throw in benzamidine (1mM) for good measure - possibly over cautious but it doesn't hurt. If your protease inhibitor cocktail has EDTA added, this may have an effect as it could strip off an important cation needed for acivity? Ultimately, it would seem likely your mutation/variant is genuinely less active - is this aa change in the active domain? Sounds like you may be able to publish this if it's a novel observation (and clinically relevant), so good luck with with writing up the manuscript!
As others have noted it is possible that it effects enzyme activity because of its inhibition of proteases. In addition, protesaes and esterases share a similar mechanism. If your protein is an esterase or a protease PMSF may be inhibiting it. Also keep in mind that PMSF stock solutions are dissolved in ethanol or IPA. Depending on your stock concentration and your final concentration you may be putting in too much ethanol or IPA. I have never had a problem with this because I use 200mM stocks and 1mM final concentrations. At this point I am grasping at straws. The most likely explanation is that your mutant has less activity.
I think you can add some PMSF when you purify your WT, then test activity to see whether it can affect enzyme activity
People usually add PMSF to the culture medium when proteolysis is the main concern. However, you can get control on this process by adjusting the pH of the medium. If your recombinant enzyme is susceptible to PSMF you should try to control the pH of the medium instead using this protease inhibitor.
Good luck.
Perform controls and use same condition when conducting such analysis will be very beneficial;
Also do some background check on your protein target will give you ideas of factors may affect its activity;
It's unlikely that PMSF inhibits the mutant because of it's lack of effect on the WT but it's ovious to omit PMSF in the preperation and determine acttivity. The comment that the serine residue has a drastically altered pK is incorrect because the reaction with the serine OH represents general base catalysis.
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