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Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain.

Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.

ABSTRACT

The regular surface protein array (S layer) present on Aeromonas hydrophila TF7 is composed of a single species of protein of apparent molecular weight 52,000. This protein was extracted from whole cells by treatment with 0.2 M glycine hydrochloride (pH 3.0). The protein was purified to homogeneity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Amino acid composition analysis showed that the protein contained 520 residues per molecule, 41% of which were hydrophobic. Cysteine was absent. A pI of 4.6 was determined for the protein, and only a single isoelectric form was detected. The purified protein displayed the hydrophobic characteristic of binding to octyl-Sepharose gels, but the salt aggregation test showed that it did not confer hydrophobicity to the cell surface when present as an intact S layer. The molecule aggregated strongly in aqueous solution as determined by sedimentation equilibrium studies. Circular dichroism spectra showed that the S-layer protein was composed of a large amount of beta-sheet (approximately 44%), a limited amount of alpha-helix (19%), and 12% beta-turn, with the remainder of the molecule being aperiodic. No significant difference in secondary structure content was measured in the presence of the metal chelator EDTA. The N-terminal amino acid sequence was determined for the first 30 residues. No sequence homology with other S-layer proteins was found.

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