A NOVEL ACTIVITY OF OMPT - PROTEOLYSIS UNDER EXTREME DENATURING CONDITIONS

A novel property of the bacterial outer membrane protein T, OmpT, has been discovered. It is active under extreme denaturing conditions. Thi s finding emerged during characterization of a protease associated wit h the degradation of recombinant proteins expressed as inclusion bodie s in Escherichia coli. These inclusion body proteins are stable to pro teolytic degradation until they are solubilized by denaturation. The p rotease 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 Om pT. A previously unknown property of this enzyme, OmpT's preference fo r denatured substrates, may provide a clue to its physiological functi on. To facilitate further characterization of this proteolytic activit y, we have optimized a system to extract and assay OmpT under denaturi ng conditions using a soluble substrate, rabbit muscle creatine.