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

Abstract

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.

Structural Basis for the Kexin-like Serine Protease from Aeromonas sobria as Sepsis-causing Factor

“…Analysis of Nicked ASP Using Mass Spectrometry-In earlier studies, we purified ASP through a series of successive column chromatography steps using a DEAE-Sephadex A-25 column, a hydroxyapatite column, and a gel filtration column in an HPLC system (12). Because that method was both laborious and time-consuming, we have developed an improved method that entailed expressing recombinant ASP in A. sobria T94, a strain we found to produce large amounts of ASP.…”

“…Purification of Nicked ASP-To purify ASP, A. sobria strain T94 harboring pSA19 -5528 was cultured in Luria broth supplemented with chloramphenicol (50 g/ml) for 20 h at 37°C with shaking (12). After cultivation, the cells were removed from the culture fluid by centrifugation, and ammonium sulfate was added to the supernatant to 50% saturation.…”

“…Crystallization-We purified ASP from the culture supernatant of the A. sobria T94 strain transformed with pSA19-5528, including the asp and orf2 genes as described previously (12), and its sequence has been already registered in GenBank under the accession number AF253471. The sitting drop vapor diffusion method with polyethylene glycol (PEG) was used to prepare ASP crystals, which grew in 3-4 days at a PEG concentration of 10%.…”

Salt in Surroundings Influences the Production of Serine Protease into Milieu by Aeromonas sobria

“…We have already shown that ASP matures through a peculiar maturation pathway (24), illustrated in Fig. 9A.…”

“…9A. Briefly, two proteins are encoded in the operon of the asp gene, ASP and ORF2 protein, which are located at the 5' and 3' ends of the operon, respectively (24). Both ASP and ORF2 protein move into the periplasmic space and associate in the space.…”