I'm struggling to purify a full-length His-tagged recombinant protein with GFP domain and S-tag.
I produced the protein in E.coli batch culture and purified it using Ni-NTA affinity agarose beads from QIAGEN where I incubated the beads with the protein overnight at 4° then used gradient imidazole to elute the protein. I performed a WB using Abs against the His-tag, GFP, and S-tag where I could see only fractions of the protein and never the full length!!
Can anyone provide insight? would love to know your suggestions.
Dear Rahaf,
It looks like you are getting proteolyusis of your construct. One thing would be to add a protease inhibitor cocktail (EDTA-free, of course) to your extraction buffer. However proteolysis most likely occurs in vivo, which is a common problem, particularly with multidomain proteins. You can try to mitigate it by performing expression at low temperature, e.g. 14°C overnight.
Dear Rahaf,
I agree with Pierre. Also, have you checked if the protein has no-paired cysteines? If so, you can add 1mM DTT for example to reduce those cysteines and mitigate the proteolysis produced by instability.
Many thanks,
I recommend that you avoid the overnight incubation, which gives a lot of time for proteolysis to occur. Binding of His-tagged proteins to Ni-NTA affinity resin is rapid, so one hour should be long enough. The best method is to pour the resin into a column and flow the extract over it.
Hello :)
I hope that I can still be of help here. The other are correct in their suggestions, I just want to build on them.
The use of DTT and protease inhibitors can help you.However, if you do that I would not use Ni-NTA anymore. As it is DTT sensitive. Also you are limited to EDTA free protease inhibitors.
By using INDIGO-Ni you can ignore these constrictions. As its tolerances for both DTT and EDTA are at 20 mM. It still uses the same buffers and protocols as Ni-NTA. Only the moledule binding the Nickel Ion is different. But that helps a lot.
https://cube-biotech.com/indigo-features
I agree, with what has been said above
1. avoid DTT and other reducing agents
2. use protease inhibitors, EDTA free
3. do NOT bind over night. once bound and washed, you may leave the protein on the column for prolonged time. but not before wahsing.
things should look better after following theses points
I agree with the previous comments:
1. Avoid overnight incubation, I would use 2h 4º degrees.
2. Use protease inhibitors.
3. Avoid reducing agents.
4. I'm using Ni-NTA affinity columns from QIAGEN and work pretty well, in case you need to test another system.
I agree, avoid O/N incubation. My suggestion is to try different incubation time and sure at low temp (cold room) would be the best.
I think you should avoid incubating your protein with the nickel beads for such a long time as it will make proteolysis. also use protease inhibitor cocktail. I usually make nickel bead column and after equilibrating it, load the lysate on to the column and keep it still for 30 minutes. After 30-40 minutes I start the flow at a rate of 0.5ml/minute. Then elute the protein with gradients of imidazole.
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