I am working on Mus musculus protein of 85 KD which expresses very well but I am getting degradation band so for confirmation I did Mass Spec and western blot so its degrading from the C terminus and I am getting three degradation bands at 85kd, 43 kd and 25 kd. To prevent this I have added a strep tag at the C terminus and his tag at N terminus and purified with double tag but still I am getting same pattern. Please can you suggest some good ideas?
The obvious response, which you may already have tried, is to include a cocktail of protease inhibitors in your purification procedure. You can purchase tablets that can be dissolved in the purification buffers immediately before use. They contain several different protease inhibitors.
Do the purification at 4oC, and work through it as quickly as you can to minimize proteolysis. Do not leave fractions or purified protein unfrozen for long periods of time.
If proteolysis is occurring during expression of the protein, you may be able to switch to a reduced-protease cell line/strain.
There are few things to be confirmed first:
Check the pH stability of the protein,
Check the temperature stability of the protein
Then try to extract your protein at that conditions. Hope this answers your query?
If not, please revert back.
The obvious response, which you may already have tried, is to include a cocktail of protease inhibitors in your purification procedure. You can purchase tablets that can be dissolved in the purification buffers immediately before use. They contain several different protease inhibitors.
Do the purification at 4oC, and work through it as quickly as you can to minimize proteolysis. Do not leave fractions or purified protein unfrozen for long periods of time.
If proteolysis is occurring during expression of the protein, you may be able to switch to a reduced-protease cell line/strain.
Hi there,
Your protein may contain very sensitive protease cleavage sites. Identifying (mass spec p fingerprint from the degradation bands) and then mutating these sites may be an option to get the full length protein.
Try optimal temperature, pH. If nothing work well, try to make different constructs by removing the the C terminus
The best will be off course to use protease inhibitor cocktails, which you might have already used. In case of protein instability like this, i would pay very much attention to pH and temperature. maybe fusing with a larger tag like GST can help. you can introduce a thrombin cleavage site, so that later if necessary, you can cleave off the tag.
Along with above all mentioned possibilities, one reason might be the tendency of protein to get cleaved. There are plenty of online servers that could tell you if your protein has tendency to undergo auto cleavage. Also now a days many E coli strains are without proteases, so I am not sure if inhibitor cocktail would be of much use. Check for autolytic tendency or else check stability
If you need advice beyond that already offered I would outline in detail the expression system that you are using, whether the protein is intracellular or secreted, and if degradation is visible within immediate crude extracts. i.e to determine if degradation is occurring during expression or purification.
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