Figure 2.

A) Western blot analysis to evaluate the expression of the mutant CDA Y33G in presence of specific ligands of cytidine deaminase in the culture medium.

wt: wild-type CDA (positive control); neg. contr: no CDA ligands; st: Bio-Rad low molecular weight standard.

B) Western blot analysis to evaluate the presence of inclusion bodies in the crude extracts of Y33G. 1: no glycerol in the culture medium. E. coli cells were lysed and after centrifugation the supernatant was resuspended in buffer A without glycerol and analyzed; 2 and 4: no glycerol in the culture medium. E. coli cells were lysed and after centrifugation the pellet was resuspended in 8M urea and analyzed; 3: no glycerol in the culture medium. E. coli cells were lysed and after centrifugation the supernatant was resuspended in buffer A with10 % glycerol and analyzed; 5: 10% glycerol in the culture medium. E. coli cells were lysed and after centrifugation the supernatant was resuspended in buffer A with 10 % glycerol and analyzed; 6: 10% glycerol in the culture medium. E. coli cells were lysed and after centrifugation the pellet was resuspended in 8M urea and analyzed. St: Bio-Rad low molecular weight standard