Urea is a small organic molecule whose structure will not be affected by the presence of proteins. However, it is a strong caotrope that affects protein folding. Cosmotropes (sugars, glycerol, amino acids...) interact with water, thereby reduce the available water concentration and hence the mobility of protein segments against each other. This stabilises the tertiary structure of proteins, which is the reason that such compounds are used for example in protein crystallisation. Caotropes (urea, thiocyanate, guanidinium), on the other hand, react not so much with water, but with the proteins. This interaction competes with the non-covalent bonds that hold the tertiary and quaternary structure of proteins together, destabilising them. They are used to stop enzymatic reactions, or to unfold proteins for example in electrophoresis.

A list of compounds going from strong cosmotropic to strong caotropic is called Hofmeister series (doi:10.1007/BF01838161).

Urea is a small organic molecule whose structure will not be affected by the presence of proteins. However, it is a strong caotrope that affects protein folding. Cosmotropes (sugars, glycerol, amino acids...) interact with water, thereby reduce the available water concentration and hence the mobility of protein segments against each other. This stabilises the tertiary structure of proteins, which is the reason that such compounds are used for example in protein crystallisation. Caotropes (urea, thiocyanate, guanidinium), on the other hand, react not so much with water, but with the proteins. This interaction competes with the non-covalent bonds that hold the tertiary and quaternary structure of proteins together, destabilising them. They are used to stop enzymatic reactions, or to unfold proteins for example in electrophoresis.

A list of compounds going from strong cosmotropic to strong caotropic is called Hofmeister series (doi:10.1007/BF01838161).

I agree with Dr. Engelbert answer

In other words, 6M urea inhibits the activity of lysozyme by denaturing (unfolding) it.

Urea is a denaturant and will unfold proteins, but the extent of denaturation depends on the protein and urea concentration. Some proteins can maintain activity in high concentrations of urea. You wouldn't know unless you did either an activity assay at varying concentrations of urea or a denaturation curve, but for what you say you're doing it's not worth doing that. For what you're doing to extract a protein, I would recommend to treat your cell pellets with lysozyme first for some amount of time and then add the urea.

If you are trying to purify a protein in an active state from your bacterial cell pellets, do not use urea. If you don't need or want the proteins to be active and folded, you can use urea (after lysozyme treatment to break down the cell walls, as Arden Doerner said).

In order to retain protein activity, try to use native lysis conditions that avoid urea or guanidinium.

Urea alone can't do the confirmational changes in the lysozyme/ or protein . Normally inhibitor sugars bind to the n-acetyl site to contribute to the confirmational changes. I suggest you to read the following article:

Article A structural basis for the interaction of urea and lysozyme

Thank you very much for everything, your comments are very enriching.

I'm trying to extract a recombinant protein of BL21 DE3 cells with the pET 32B vector, however the proteins are always degraded, we use Lysozyme, EDTA, UREA, Protease inhibitors, PMSF and we left it treating the pellet overnight. the next day We performed thermal shock 3 times at -80 ° C 15 min and 4 ° C 15 min. This pellet was induced 4 hours with 1mM IPTG.

however, we noticed a clibaje of the protein and we did not manage to obtain sufficient for the purification.

If anyone can please recommend any protocol in these cells and with this vector, I would be very grateful. Thank you all for your comments and help.

You need to work on the extraction buffer !!

May be your lysozyme and protease inhibitors playing a role to degrade your protein of interest.

Do not treat your pellet overnight. That leaves much time for degradation. See if you can find either a probe-type sonicator or a French Press. Once you have the cell pellet, you can either freeze it at -80oC or treat it immediately. Resuspend the cell pellet with a buffer to adjust the pH, such as 50 mM HEPES pH 7.5. Include the protease inhibitors, DNase + MgCl2, and RNase, but not EDTA or urea. Break the cells by French Press or sonication, keeping the extract ice-cold at all times. Proceed immediately to the first purification step once the cells are broken.

• A French press is exactly what I would use, but with sonifiers my experience is "mixed".
• It is important to get a protease-free nuclease, http://www.worthington-biochem.com is a reliable source (no affiliation).
• Remember that PMSF decomposes within 20 min in aqueous solution, add immediately before use from a 1 M stock in an aprotic solvent like i-propanol. Do not use DMSO, which can penetrate intact skin and take dissolved substances with it. You need it only during lysis as it inactivates rather than inhibits proteases.

Hi!

I think you should use neither urea Nor Lysozyme for protein study as

1)When you use lysozyme for lysis of bacteria and try to isolate bacterial protein; it become very hard to remove lysozyme from the mixture.

2)Urea denatures protein so it will not be a good idea for protein isolation.

You may try other lysis procedure like sonication or sucrose lysis buffer or TRI reagent.

I recommend you to use TRI reagent (Sigma-Aldrich) protocol for the same.

GOOD LUCK

TRI reagent will denature the proteins completely.