Study on Extracelluar Secretion and Folding Mechanism of a Thermophilic Protease (Aqualysin I)

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The precursor of aqualysin I, an extracellular protease produced by Thermus aguaticus, consists of four domains : an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a Cterminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed on treatment at 65ﾟC. In this research project, roles of the pro-sequences in folding of the precursor and extracellular secretion of aqualysin I were studied and several findings were obtained, as follows.1. The N-terminal pro-sequence (113 amino-acid residues) is required for the formation of stable conformation of the precursor and the production of active aqualysin I.2. Complete deletion of the C-terminal pro-sequence (105 aminoacid residues) does not affect the production of active enzyme, indicating that the C-terminal pro-sequence is not essential.3. A non-covalent N-terminal pro-region aids the folding of the protease domain and the production of active enzyme, just as a molecular chaperon. In this case, the C-terminal pro-sequence is rather inhibitory as to its production.4. The C-terminal pro-sequence is required for a 38-kDa protein (a precursor of the mature enzyme with the C-terminal pro-sequence) to be bound to the membrane fraction (probably outer membrane fraction).5. Expression system of the aqualysin I gene is constructed using a host-vector system for Thermus thermophilus, and correctly processed aqualysin I is secreted into t e culture medium.6. In the expression system of T. thermophilus, the C-terminal pro-sequence is essential for extracellular secretion of aqualysin I.