Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain.
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.
Dooley
J S
JS
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
McCubbin
W D
WD
Kay
C M
CM
Trust
T J
TJ
D000333:Aeromonas / Q000032:analysis*; D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification*; D002942:Circular Dichroism; D007525:Isoelectric Focusing; D008562:Membrane Glycoproteins / Q000032:analysis / Q000302:isolation & purification*; D011487:Protein Conformation
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C489049:regular S-layer protein, bacteria