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Cloning, characterization, and insertional inactivation of a major extracellular serine protease gene with elastolytic activity from Aeromonas hydrophila
Author(s) -
Cascón A,
Fregeneda J,
Aller M,
Yugueros J,
Temprano A,
Hernanz C,
Sánchez M,
RodríguezAparicio L.,
Naharro G.
Publication year - 2000
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1046/j.1365-2761.2000.00206.x
Subject(s) - tmprss6 , biology , protease , biochemistry , serine protease , peptide sequence , masp1 , proteases , aeromonas salmonicida , microbiology and biotechnology , amino acid , aeromonas hydrophila , nucleic acid sequence , signal peptide , molecular cloning , gene , enzyme , bacteria , genetics
The gene ahpA from Aeromonas hydrophila AG2 encoding an extracellular serine protease, named AhpA, was cloned in pUC18 plasmid. Nucleotide sequence analysis revealed an open reading frame of 1875 bp encoding a 625 amino‐acid protein with a molecular weight of 67 567 Da. The gene ahpA was efficiently expressed in Escherichia coli C600 and in the non‐proteolytic A. salmonicida masoucida , which was able to overproduce the 64‐kDa protease found in the culture supernatant. The N‐terminal amino acid sequence of the purified protein revealed a perfect match with the deduced DNA sequence starting at AAT (Asn‐25), indicating that AhpA is synthesized as a pre‐enzyme with a 24‐amino‐acid signal peptide and a 601‐amino‐acid mature extracellular protease. Purified protease had an optimum pH of 7.5 and its activity was strongly inhibited by PMSF, a serine protease inhibitor. The protease hydrolysed casein and elastin. The amino acid sequence of AhpA was highly homologous to A. salmonicida serine protease AspA. Inoculation of A. hydrophila ahpA mutant into trout suggests that the major AhpA secreted protease is not essential for virulence.