M A R C B L

mains that are connected by a long cytoplasmic loop. The protease substrate can be engineered into the cytoplasmic loop of TET without loosing TET function. Cleavage of TET by the protease that is also heterologously expressed in the same strain makes the E. coli sensitive to tetracycline [38,39]. An inhibitor of the protease allows the E. coli to grow in the presence of tetracycline as the TET protein remains intact.

Another more intricate system has been developed in E. coli for identifying protease inhibitors. This system depends upon the phenomenon that a mutation takes place in the S12 ribosomal protein of the 30S ribosomal subunit that makes E. coli resistant to streptomycin [40]. Such a mutation in the S-12 ribosomal protein is simulated by making a S-12 fusion protein with the protease substrate. The E. coli expressing the S-12-peptide-substrate chimeric protein is resistant to streptomycin. When the protease is expressed in the same E. coli expressing the S12-peptide-substrate chimera, the substrate is cleaved from S-12 and the E. coli becomes sensitive to streptomycin. Protease inhibitors preserve the S12-peptide-substrate chimera, and the E. coli remain streptomycin-resistant (Fig. 7). This screening system is notable because protease-dependent, dominant phenotypes are more sensitive than recessive phenotypes [40].

Figure 7 A bacterial protease system. A mutation in the S12 protein in the 30S ribo-somal subunit makes E. coli streptomycin resistant. A chimeric S12-protease substrate protein mimics a mutation, and E. coli expressing S12-protease substrate constructs are streptomycin resistant. When the test protease is coexpressed in the streptomycin resistant E. coli, the protease substrate is cleaved from S12 and the E. coli reverts to streptomycin sensitivity. Protease inhibitors would prevent the regaining of streptomycin sensitivity.

Figure 7 A bacterial protease system. A mutation in the S12 protein in the 30S ribo-somal subunit makes E. coli streptomycin resistant. A chimeric S12-protease substrate protein mimics a mutation, and E. coli expressing S12-protease substrate constructs are streptomycin resistant. When the test protease is coexpressed in the streptomycin resistant E. coli, the protease substrate is cleaved from S12 and the E. coli reverts to streptomycin sensitivity. Protease inhibitors would prevent the regaining of streptomycin sensitivity.

Was this article helpful?

0 0

Post a comment