A 38-KDA PRECURSOR PROTEIN OF AQUALYSIN-I (A THERMOPHILIC SUBTILISIN-TYPE PROTEASE) WITH A C-TERMINAL EXTENDED SEQUENCE - ITS PURIFICATION AND IN-VITRO PROCESSING

The precursor of aqualysin I, an extracellular subtilisin-type proteas e produced by Thermus aquaticus, consists of four domains: an N-termin al signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence, In an Escherichia coli expression syst em for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulate d in the membrane fraction and processed to a 28 kDa mature enzyme upo n heat treatment at 65 degrees C, The 38 kDa precursor protein is sepa rated as a soluble form from denatured E. coli proteins after heat tre atment, Accordingly, purification of the 38 kDa pro-aqualysin I was pe rformed using chromatography, The purified precursor protein gave a si ngle band on SDS-polyacrylamide gels, The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The puri fied precursor protein was processed to the mature enzyme upon heat tr eatment, The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqual ysin I or upon an increase in the concentration of the precursor, sugg esting that the cleavage of the C-terminal extended sequence occurs th rough an intermolecular self-processing mechanism.