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Proteolysis, Instability or Truncated Expression


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#1 dcch

dcch

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Posted 31 December 2010 - 08:37 PM

Dear forumer,

I am dealing with a hypothetical protein. Previously, heterogenous expression of this protein in E.coli was very hard. It has been expressed as His-tag and GST-tag fusion protein, as a total insoluble protein. Then, I tried to express the protein with MBP-His-tag fusion. Finally I got quite a lot of soluble protein. This is a great news :lol:. However, when I perform purification, I always get multiple bands, ranging from 40 to 82 kDa. :(

Personally, I suspected it is trancated protein or might due to instability of the protein. I do not think this is non-specific binding of contaminant. It is because the multiple bands only present in between 40 to 82 kDa (Size of MBP + my protein) but not in other size. Protease inhibitors cocktail has been added during purification steps.


Any idea or suggestion to improve this condition?

Thanks a lot!

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