I have tried the purification of human recombinant interferon beta-1b from E. coli. However, I'm not able to solubilize the inclusion bodies formed in this case. Please give suitable suggestions in this regard or if anyone has worked on this protein.
I've tried {Tris (30 mM/50 mM) + NaCl (150mM)+ 2-5% glycerol+ 2% sarcosine} and {0.5 % SDS alongwith Tris+NaCl}. I even tried with 6M urea.
After preparing the IBs, i have resuspended and stored it in ddH20 at -20. During solubilization, one vial(1 ml) of IB's were thawed and mixed in 40ml of solubilization buffer, kept on rocker shaker at 25 degrees. I even tried it at 37 degrees. After solubilization, the solution turned out to be viscous and transparent.
I would use Tris pH 8+8 M urea. If this doesn't work, try 4-6 M GuaHCl. Since it's a secreted protein, I suppose it has disulfide bonds? In that case, add 30 mM DTT to the solubilization buffer, and add a redox pair such as cysteine/ cystine or GST/GSSG to the refolding buffer. Concentration of redox pair can be varied to optimize recovery, but a good starting point would be 1 mM.
Good luck!
Yeah, I tried with 8M urea and 6M GdmCl. The solution turns viscous and sticky after a few hours. It blocked the Ni-NTA column and did not bind to it.
Currently, I am facing another issue with the interferon beta protein expression. There's one bacterial protein expressing in high amount and it is hindering the expression of interferon. Please give me suggestions on this also. I used BL21 DE3 cells for the expression.
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