A novel activity of OmpT. Proteolysis under extreme denaturing conditions

Abstract

A novel property of the bacterial outer membrane protein T, OmpT, has been discovered. It is active under extreme denaturing conditions. This finding emerged during characterization of a protease associated with the degradation of recombinant proteins expressed as inclusion bodies in Escherichia coli. These inclusion body proteins are stable to proteolytic degradation until they are solubilized by denaturation. The protease that degrades them under denaturing conditions was identified as OmpT on the basis of substrate specificity, inhibitor profile, and confirmation that its N-terminal sequence is identical with that of OmpT. A previously unknown property of this enzyme, OmpT's preference for denatured substrates, may provide a clue to its physiological function. To facilitate further characterization of this proteolytic activity, we have optimized a system to extract and assay OmpT under denaturing conditions using a soluble substrate, rabbit muscle creatine kinase.