Dear all,
I am expressing in BL21(DE3) E. coli cells and subsequently purifying a 6x-His-tagged 50 kDa truncated protein which looks like to be very unstable. Its domains are literally falling apart right after cell lysis giving huge 30 kDa and 20 kDa degradation product bands on the gel I'm running after the first purification step (His-tag chromatography).
Even though I am adding anti-protease tablets before lysing the cells (5-15 seconds on-off, 50% amplitude for 10 mins) and 1 mM PMSF in the purification buffers I cannot prevent its degradation and the yield of intact protein I'm getting at the end is really poor.
Lysis/His-tag chromatography buffer
20 mM Tris-HCl pH 7.5
150 mM NaCl
20 mM imidazole
1 mM DTT
0.1% Tween-20
His-tag chromatography elution buffer
20 mM Tris-HCl pH 7.5
150 mM NaCl
300 mM imidazole
1 mM DTT
Gel filtration chromatography buffer
20 mM Tris-HCl pH 7.5
150 mM NaCl
1 mM DTT
1 mM PMSF
I've tried to monitor the expression of the protein before and after its induction overnight. It looks like the protein is not degraded until it's within the bacteria.
Is there anything I can change in my buffers or in the whole process (I'm already performing all the steps at 4ºC) to slow down the degradation of the protein?
Thank you in advance for any recommendation you can give me
Hi there,
What do you mean by : " It looks like the protein is not degraded until it's within the bacteria"?
Hi @Dominique, I mean that in the sds page I ran after the protein expression was induced I haven't any degradation product.
There may be a protease hypersensitive amino acid sequence linking the 30 and 20 kDa domains. The protein may not actually fall apart if there are interactions between the two domains, but since SDS-PAGE denatures the protein, you see it as two separate bands. In any case, if you can find out where the cleavage is occurring, you may be able to introduce a mutation to thwart the proteolysis. Another option is to switch expression strains to one with less protease activity (although BL21 is already deficient in this Lon and OmpT proteases).
Hi @Adam, after protein expression induction SDS-PAGE showed the presence of the 50 kDa intact protein alone. In any of the following purification steps the 50 kDa band is always present but is coming together with the degradation products mentioned above.
Leonardo Feletto ,
How is the protein obtained? As inclusion bodies, or in soluble form? Also, what anti-protease tablets are you using exactly?
Hi @Alejandro, the protein is soluble. I'm using cOmplete, EDTA-free protease inhibitor cocktail tablets
Hi there,
You might increase salt concentration up in your lysis buffer as well as washing buffers to see if the phenomenon still happens?
Hi Leonardo
I am routinely doing these kind of purification, and and normally use higher ionic strenght either in the binding or elution buffer of the metal-chelating chromatography. My default buffer composition contains 1 M NaCl either in the binding or in the elution buffer. Higher ionic strenght will help the purification yield and decrease degradation at least in theory. In consequence I agree with Van comments.
Best of Luck. Paco
Dear Francisco J. Enguita and Van Dung Pham unfortunately this protein is really tricky. I came to the conditions I described after trying purifications at 300 mM NaCl -- at this ionic strength the protein is behaving weirdly, forming very large soluble aggregates that are coming out right after the void volume in the gel filtration chromatography column... In any case even at 300 mM salt it was possible to observe the formation of degradation products. Maybe the answer is to find a salt concentration in between (e.g. 200 mM?)
Leonardo Feletto ,
If the degradation takes place only during/after cell lysis then it is likely caused by a cell envelope- or periplasm-associated protease; most probably a metalloprotease. Have you tried breaking the cells in the presence of Complete+EDTA to see what happens? I know you won't be able to use IMAC right away if you do, but if EDTA halts degradation, then you have a pointer on how you should start to modify the purification procedure (you could do ammonium sulphate cuts, for instance, or use the new Indigo Ni-agarose resin from Cube Biotech which is supposedly resistant to chelators and reducing agents -haven't used it myself, just saw someone raving about it).
Hope it helps!
Dear Leonardo
2 questions:
1) Why did you add the DTT? Is your protein contains S-S bridge?
Becauseb can have a double role, can be good for some proteins which contains Cys not involved in S-S but can induce unfolding and therefore increase the protein susceptibility to proteases in case that your protein contain structural S-S bonds
2) Sonication 10' at 50%?Did you check if the sample temperature increase? Generally i prefer a weaker sonication approach as 30-40%, 10 cicle 30''ON/2' off in ice or something like this because is it important that sample temperature remain low not only because high temperature can induce unfolding but also because the protease are more active at high temperatures.
good luck
ciao
Manuele
Dear Alejandro Martin , thank you for your suggestions! I've never tried Complete+EDTA protease inhibitors.
Dear Manuele Martinelli ,
1) my protein doesn't have S-S bridges and I am using reducing agents by default to keep Cys residues reduced... I haven't ever performed a purification of this protein without DTT, so maybe it's something I can try.
2) I'm always sonicating my cells in ice, but reducing incubation time during sonication as you're suggesting, it's another thing I can try. Thank you!
- PMSF is rapidly degraded in water, half-life ~20 min. There is no point in having it in SEC buffer, where run times are hours. That is no problem, however, as PMSF is an inactivator, not an inhibitor, of Ser-proteases. Add it to the lysis buffer immediately before use. Once you killed the proteases, they are dead for good.
- Could your protein be "intrinsically disordered"? ID proteins are very difficult to purify.
- Sometimes, proteases are funny. I once had a protein that required the presence of 6-aminocaproic acid, even though PMSF was present (both supposedly act on Ser-proteases).
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