I am trying to purify a recombinant His tag fused protein from E.coli. I am facing several problems after I sonicate, centrifugate and purify it using His trap Ni column: 1- I got too much background after running SDS-PAGE. 2- My protein stick within the pellet during centrifugation. I have tried different buffers in different concentrations but always either have non pure protein or have Iost my protein. The best buffer was with 50mMTris, 500mM NaCl, 10 imidazole.
Hi Safaa,
As Shruti mentioned, the pI of your protein is very important and you should check it to see if your protein is actually stable at the pH of the buffers you are using for Ni-NTA. If your protein has a pI that is incompatible with Ni-NTA purification at pH 8.0, then you should change the pH of your buffers (you can only go down to pH 7, but over pH 7.6 is best and you shouldn't go much higher than pH 9) or change to a different purification method (GST, MBP, ion exchange, etc...).
If the pI is not the problem and since your protein seems to be mostly insoluble and you don't want to affect its conformation by purifying it from inclusion bodies, which would denature it (and yes, often proteins won't refold after denaturation), I think you should start over again.
Claus is correct and you probably should try to re-clone your gene, changing the His-tag from one end of the construct to the other might help, but in your case I would try a solubilisation tag (SUMO-tag or MBP-tag for example). These tags are small, soluble proteins that are fused to your protein of interest and help it stay in solution. They can either have a His-tag on them as well (SUMO-tag) or you can use a different column that selects for the tag itself (MBP-tag).
Also, as Patrizia and Zahir mentioned, changing the expression conditions often helps. Lowering the temperature to 30, 25 or 18oC and increasing the expression time to 12 or 24 hours often helps with protein stability. Also, using a different growth medium (2xYT instead of LB), lowering the OD at which you induce (OD600 = 0.6 or 0.4 is often better for difficult proteins) and the IPTG concentration (use 0.1 to 0.5 mM instead of 1 mM) can help.
Lastly, some proteins express better in some E. coli strains than in others, particularly if your gene of interest comes from an organism that has rare codons, in which case you'll need to use strains that can code for these codons (e.g. Rosetta can code for many rare codons that the average BL21 cells can't).
Before sonication, add 50mMt Tris-HCl ph 8, 300mM NaCl, ImMDTT, ImM PMSF, 0.5%Triton-X100 and 10% Glycerol and then do sonication for atleast two hours. Then centrifuge at 13,000g for 10 minutes and remove the pellet. Filter the lysate through 0.45 u filter and add the clear lysate to the Ni column and incubate for 2-3 hrs. Aftre incubation, start washing with 10mM imidazole, followed by 20, 40, 80, 150, 200 and 250 mM imidazole. Do the SDS for your purified fraction. I do hope it works well in almost all cases.
Hi! If you find your protein with your pellet, I guess it's not so much soluble. You can try different expression conditions to increase its solubility, e.g. different IPTG concentrations, different temperatures, different E.coli strains...little changing in these parameters can usually make a great difference.
Thanks for your help! I want the fused proteins (PI 6) for the reaction with electrophiles. I don`t want to lose protein conformation. The big problem is that my protein stick with the pellets during centrifuigation, and any change in sonication time, potential, buffers PH 8 , or use 0.45 u filter or triton x affect on protein yield! I am afraid of using urea or others might affect the protein conformation?
Did you check the calibration of your columns before usage with different imidazole concentrations?
Also the His-Tag might be inappropriate for your protein, since it might be not/insufficiently exposed.
What about membrane association?
Thanks again! Claus Georg Gustav Schöning, Check the calibration of your columns? membrane association! may be.
Shruti Sharma, so it is inclusion body?
Other suggestions include: Culture the E.Coli at16C overnight, and use KCl instead of NaCl at a concentration of 250mM. KCl is better than NaCl although has a little effect. Try to start your elution with 10mM Imidazole in PBS without any salt followed by 20 and 4omM Imidazole. Then add KCl salt to the above concentration, 50mM Tris-HCl, 20 mMHEPES. Then do dialysis to remove imidazole and then do IE. If you dont want salt, use desalting column before IE. After this run SDS to check your protein. or you can directly use size exclusion column according to the expected size of your protein.Do remember to use the proper buffer in elution of IE which could work best in your downstream applications.
Hi Safaa,
As Shruti mentioned, the pI of your protein is very important and you should check it to see if your protein is actually stable at the pH of the buffers you are using for Ni-NTA. If your protein has a pI that is incompatible with Ni-NTA purification at pH 8.0, then you should change the pH of your buffers (you can only go down to pH 7, but over pH 7.6 is best and you shouldn't go much higher than pH 9) or change to a different purification method (GST, MBP, ion exchange, etc...).
If the pI is not the problem and since your protein seems to be mostly insoluble and you don't want to affect its conformation by purifying it from inclusion bodies, which would denature it (and yes, often proteins won't refold after denaturation), I think you should start over again.
Claus is correct and you probably should try to re-clone your gene, changing the His-tag from one end of the construct to the other might help, but in your case I would try a solubilisation tag (SUMO-tag or MBP-tag for example). These tags are small, soluble proteins that are fused to your protein of interest and help it stay in solution. They can either have a His-tag on them as well (SUMO-tag) or you can use a different column that selects for the tag itself (MBP-tag).
Also, as Patrizia and Zahir mentioned, changing the expression conditions often helps. Lowering the temperature to 30, 25 or 18oC and increasing the expression time to 12 or 24 hours often helps with protein stability. Also, using a different growth medium (2xYT instead of LB), lowering the OD at which you induce (OD600 = 0.6 or 0.4 is often better for difficult proteins) and the IPTG concentration (use 0.1 to 0.5 mM instead of 1 mM) can help.
Lastly, some proteins express better in some E. coli strains than in others, particularly if your gene of interest comes from an organism that has rare codons, in which case you'll need to use strains that can code for these codons (e.g. Rosetta can code for many rare codons that the average BL21 cells can't).
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