Protease I from Pyrococcus furiosus
Publisher Summary Pyrococcus furiosus is a hyperthermophilic archaeon from the order Thermococcales capable of growth on a variety of proteinaceous and carbohydrate-containing substrates. Analysis of gelatin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) gels indicates that at least 11 endoproteinases are active in the cell extracts of this organism 2'3 and the following proteases have been characterized: protease I (PfpI), pyrolysin, proteasome, prolyl oligopeptidase, and proline dipeptidase. The gene encoding the 19-kDa subunit of PfpI has homologs in nearly every organism and cell examined to date, ranging from Escherichia coli to Homo sapiens; this ubiquity and evolutionary conservation indicates that it may play a fundamental physiological role. Efforts to study this issue have been exacerbated by difficulties encountered in obtaining significant amounts of a particular assembly of PfpI in either a native or a recombinant form. Native PfpI undergoes autoproteolysis and/or disassembly during direct purification from P. furiosus biomass and exists in multiple (singleto multisubunit) forms in vitro. The production of a recombinant form of PfpI is also problematic due to its toxicity toward mesophilic hosts. This chapter describes several methods that have been used to purify PfpI directly from P. Furiosus cell extracts are described here, together with an assay to detect proteolytic activity, a procedure to determine its molecular mass, and approaches to minimize PfpI-catalyzed proteolysis of other P. furious proteins.