What causes loweryields for eucaryotic proteins using the IMPACT system in E.coli?
40% of the eucaryotic 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 eucaryotic protein purification using the IMPACT System can be attributed to the fact that many eucaryotic proteins express poorly 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 procaryotic and eucaryotic 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 eucaryotic expression system may increase expression levels of eucaryotic proteins. Stratagene sells BL21-CodonPlus Competent Cells. These cells are engineered to contain extra copies of
