Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escheri chia coli. This genetic test is based on the specific proteolysis of a sign aling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a mode l system we used the human immunodeficiency virus (HIV) protease. When an H IV protease processing site, p5, was inserted in frame into the AC polypept ide, the resulting ACp5 protein retained enzymatic activity and, when expre ssed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV prote ase coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the result ing AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya- phenotype. In the presence of th e protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cel ls were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could rep resent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients und ergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.