Fig. (2). Evaluation of Sites for Circular Permutation A. Cleavage dependent activation of CP-Luc/ENLYFQS fusion protein. Cell-free translation reactions were diluted 1:1 in 2X TEV protease buffer, incubated at 30°C for 30 minutes ± 10 U TEV protease and luminescence was measured from 5 µL aliquots. Data are the mean values, N = 3. Error bars are the standard deviation of the mean. B. Five µL of the protease digest reaction was size-fractionated on NuPAGE^® gels and FluoroTect^TM labeled proteins were visualized on a fluoroimager. TEV protease digestion resulted in the expected sized fragments. Note, the digestions were not complete, and therefore, residual undigested 60 kDa protein remains in all of the “+” lanes. No expressed protein is visible in the no DNA lanes.