Expression of IL-4, GM-CSF in E.coli

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juichin
juichin's picture
Expression of IL-4, GM-CSF in E.coli

Hello,

I tried to express His-tagged IL-4, IL-13 and GM-CSF in E.coli. These proteins mainly accumulated as IB. During purification (after IB-purification and denaturation) proteins fragmented between His-tag and target protein.

Does anyone have any suggestions?

In addition, we are looking for a method for soluble expression of these proteins (optimize strain, vector, media, and induction conditions). IB-purification also seems to be inefficient (e.g endotoxin depletion). Does anyone have an optimize strategy for purification of IB, especially IB of cytokines and purification of renaturated cytokines preventing proteolysis?

Thanks for your efforts,

naren
naren's picture
 Dear Mkr,

 Dear Mkr,

are you getting degradation while the IBs are solubilized in chaotropic agents??

you  might aware that , in denaturing conditions and in reducing environment protease action is less and purification of his tag proteins is easier.

pushing the above said products to soluble portion is difficult. 

solubilize IBs in 6M GuHCL and in 0.5 mM EDTA and 2 mM TCEP/DTT and do the IMAC purification 

up date if you find any better thoughts good luck