Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

Abstract

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M(r) value of 57,190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27-61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein is synthesized as a 557-amino acid precursor and processed to produce a mature protein of M(r) 54,505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

“…Taken together these results suggests that p57 is autoproteolytic (Barton et al 1997). Sequence analysis of p57 is consistent with this possibility as there is homology between the second repeated region of p57 and both trypsin-like and V8 staphylococcal-type serine proteases (Chien et al 1992). In addition, a consensus serine protease motif G-X-S-X-G has been located from Gly-406 and Gly-4 10 between repeats B2 and B3 ( Fig.…”

“…P57 was partially purified and eluted with a M, of 46 500 consistent with a monomeric structure. The estimated relative molecular mass was smaller than the molecular mass of 54 505 Da calculated from the amino acid sequence of the mature protein (Chien et al 1992). The reason for this discrepancy is unknown.…”

“…Rainbow trout Oncorhynchus mykiss were 557 amino acid protein containing 2 copies of a n 81 held in 12°C well water at the Oregon State University residue direct repeat, which are 60% homologous, and Salmon Disease Laboratory and were fed Oregon 5 copies of a second 25 residue imperfect repeat Moist Pellets (Bioproducts, Warrington, OR). Samples (Chien et al 1992). One function of p57 may be to of infected ludney tissue were obtained from spawning mediate adherence of the bacteria to host tissues.…”

Production of the major soluble antigen of Renibacterium salmoninarum in Escherichia coli K12

“…Recent studies have demonstrated that the proteolytic degradation of p57 generates most, if not all, of the immunoreactive components of R. salmoninarum ECP and p57 may possess an autoproteolytic activity (Griffiths & Lynch 1991). The N-terminal sequence of p57 has been determined and gene msa encoding this protein has now been cloned and sequenced (Chien et al 1992). The aim of this study was to generate a source of recombinant p57 protein in order to remove the timeconsuming dependence upon in vitro cultures of R. salmoninal-urn.…”

“…From the sequence available for Renibacteriun~ salmoninarum gene msa (Chien et al 1992) 2 restriction sites close to the initiation codon were identified as suitable for the construction of translational fusions using the pMAL system for the production of maltosebinding protein (MBP) fusions (New England BioLabs): HpaI located at nucleotide 180 and StuI located at nucleotide 228. The HpaI truncated gene would encode a product of 513 amino acids and 49795 molecular weight, whilst the StuI truncated gene would encode 497 amino acids of 47 613 molecular weight.…”