I have a problem with the aggregation of protein connected to MBP.
My protein of interest is 18kDa protein, pI ~8.3 and with MBPtag ~60kDa and pI ~6.2
I expressed protein in E.coli BL21 cells in TB medium. Expression level Is very good.
From 30g of wet mass I am getting around 20 mg of pretty much pure (>90% purity)
protein with the tag just after one step - Dextrin-sepharose affinity chromatography.
However after LC-SEC analysis most of the protein (>80%) seems to form soluble and irreversible
aggregates.
To avoid aggregation I have tried already:
1. Keeping low protein concertation at all steps
2. Adding more glicerol 10-20% or lowering to 5%
3. Adding L-arginine to lysis and purification
4. Detergents NP-40, Triton-X100
5. Tris and Hepes buffers at pH 7.2-7.5
6. NaCl concertation in range 250-500mM
7. Different protocols of cells growth/different media etc…
8. Cleavage the tag on the resin and in the solution (because of aggregation cleavage is insufficient
and all population of protein (cleaved and uncleaved) elute together
So far nothing helps,
Do you have any idea what else I could try (I would prefer avoiding denaturation conditions since at the end I need active and properly folded protein.
Thank you in advance for your help and suggestions,
Magda
Does the precipitation occur when running SEC, as the protein elutes from the column? What buffer do you have in SEC? What buffer do you have the protein into after affinity chromatography? Do you see aggregation in this buffer?
Dear Magdalena,
I agree with Omar, you have to keep in mind and you have to trouble shoot. If your protein is eukaryotic gene then check the post translational modification site within your protein of interest if yes then go for yeast expression host or higher expression host system. To avoid denaturing you need to change the expression host system. If you want to go with the same host system you can denature your protein within the pellet then refold it with serial dilution of denaturing solution with refolding buffer. then check its integrity on SDS PAGE activity as well as activity. These are some alternates which you can try.
Thanks for suggestions!
Buffer I used for SEC was 20mMTris pH 7.4; 250mM NaCl; 5% glicerol, 0,5M arginine, 0,005%NP40, 5mMBME. Buffer after AC ws the same but included about 5 mM maltose. Protein did not precipitate in this conditions Gene codon of the protein is E.coli optimized. No special PTMs. I am thinking to use C41 or 44 strain instead BL21 maybe this will help? or high pH like even 9,5?
Thanks,
I will give a few more shots with diffrent strains of E.coli, and if will not work I will try insect cells
Dear Magdalena,
I think you are going on right path.Lets hope for the best.
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