What causes lower yields for eucaryotic proteins using the IMPACT system in E.coli ?
40% of the eukaryotic proteins tested were expressed and purified using the IMPACT System using the C-terminal fusion vectors. The final yields ranged between 0.5-1 mg/liter. The lower success rate for eukaryotic protein purification using the IMPACT System can be attributed to the fact that many eukaryotic proteins express poorly and/or as inclusion bodies in E. coli. Since the target gene is cloned into the polylinker in-frame with the N-terminus of the intein, the expression level of the fusion protein is primarily dependent on the expression of the N-terminal target protein. Thus, we would expect more variation in the yields of both prokaryotic and eukaryotic proteins than when using a C-terminal fusion system (with a highly expressed N-terminal tag). Furthermore, tests at NEB and customer feedback indicate that the use of a eukaryotic expression system may increase expression levels and solubility of eukaryotic proteins. Novagen sell Rosetta(DE3) cells while Stratagene sells BL21-
