Production of serine protease of Aeromonas sobria is controlled by the protein encoded by the gene lying adjacent to the 3' end of the protease gene

We cloned a protease gene of Aeromonas sobria and determined its nucleotide sequence. The protease is composed of 624 amino acid residues and its calculated molecular weight is 66,737.7. The amino acid sequence showed the characteristic features of a bacterial serine protease. We expressed the protease gene in Vibrio parahaemolyticus from which the synthesized protease is secreted into the culture medium as the mature form, and purified the mature protease by successive column chromatographies. The size of the mature protease is 65,000 daltons and the amino acid sequence analysis revealed that a 24-amino acid peptide at the amino terminal of the precursor is removed from the mature protease. This peptide might function as a signal peptide in translocation across the inner membrane. Subsequently, we found that the protein, designated ORF2 protein, encoded by the gene lying adjacent to the 3' end of the protease gene plays an important role in production of the protease. Mutation of the ORF2 gene did not affect transcription of the protease gene, but resulted in degradation of the protease in the cell. This shows that ORF2 protein is required for the successful production of the serine protease by cell.