Hi researchgate,
I am now attempting to purfiy some basic proteins from Streptomyces. I used E.coli as overexpression host. PI of these proteins are 7.14, 11.55 and 7.56. When I tried to purify, I used pH = 8.2 Tris based buffer but did not get anything. Do you have some suggestions on that or a recipe of purification buffer for basic proteins would be of great help.
what sorbent do you use for purification/ E. coli strain? Vector? Inductor? You provide too less information.
Dear Zhongtian Yu
you used pH 8,2 using which kind of coloumn?
ion exchange? IMAC? which coloumn?
best
Manuele
Basic proteins have high pI, meaning they are positively charged below their pI. At pH 8.2, proteins with pI of 11.55 are still strongly positively charged, and those with pI ~7.1 may be near neutral or slightly positive.
Cation exchange resins (negatively charged matrix) are the right fit, but only if pH < pI — and not too close to the pI for effective binding.
So, you likely had weak or no binding to your column at pH 8.2 because, proteins near or above their pI aren't sufficiently charged for strong ionic interaction. High pH may destabilise or aggregate some basic proteins.
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