Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis in Lutjanus erythopterus (Bloch)

• Published in: Journal of fish diseases Vol. 30; no. 8; pp. 493 - 500
• Main Authors: Cai, S.H, Wu, Z.H, Jian, J.C, Lu, Y.S
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.08.2007; Blackwell Publishing Ltd
• Subjects: activation; Amino Acid Sequence; Animals; asp gene; Base Sequence; Cloning, Molecular; DNA Primers - chemistry; Escherichia coli - genetics; Fish Diseases - microbiology; gene expression; Gene Expression Regulation, Bacterial - genetics; Gene Expression Regulation, Enzymologic - genetics; Lethal Dose 50; Lutjanus erythopterus; Molecular Sequence Data; Perciformes - microbiology; Serine Endopeptidases - analysis; Serine Endopeptidases - biosynthesis; Serine Endopeptidases - genetics; Serine Endopeptidases - toxicity; toxicity; Vibrio alginolyticus; Vibrio alginolyticus - enzymology; Vibrio alginolyticus - genetics; Vibrio Infections - microbiology; Vibrio Infections - veterinary
• ISSN: 0140-7775; 1365-2761
• DOI: 10.1111/j.1365-2761.2007.00835.x

A 750-bp internal fragment of the alkaline serine protease gene (asp) from the Vibrio alginolyticus strain HY9901 was amplified by polymerase chain reaction (PCR). The flanking sequences of the 5'- and 3'- ends of the asp gene were characterized by reverse and nested PCR. Sequence analysis showed that the asp gene contained an 1893-bp ORF encoding 630 amino acids. The deduced amino acid sequence of the ASP (alkaline serine protease) precursor showed significant homology with several bacterial alkaline serine proteases. Expression of the asp gene in Escherichia coli and activity tests of the ASP indicated that the N-signal peptide of the ASP precursor was essential to autocatalyse and fold correctly the enzyme to obtain activity. The purified ASP was lethal for Lutjanus erythopterus with an LD₅₀ of 0.25 μg protein g⁻¹ body weight.