Structural and biochemical analyses of a surface array protein of Campylobacter fetus.
Electron microscopic examination of ultrathin sections and freeze-etched and shadow cast preparations of a bovine prepuce isolate of Campylobacter fetus VC119 showed an S layer with subunits in an apparent linear arrangement. Surface radioiodination, enzyme digestion, low-pH extraction, and Western immunoblotting showed that the layer was composed mainly of one protein which is the predominant protein antigen of C. fetus. This protein was purified to homogeneity by gel filtration, ion-exchange chromatography, and high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular weight of 131,000 for this protein with a pI of 6.35, and no carbohydrate could be detected by a variety of techniques. Amino acid composition analysis showed that the protein contained approximately 1,304 residues per molecule, 41.2% of which were hydrophobic and approximately 22% of which were acidic. Cysteine and histidine were absent. Circular dichroism spectra showed that the prominent structure of the S layer protein was a beta-pleated sheet (36%) with aperiodic foldings (31%); a moderate amount of alpha-helix (28%) and a low amount of beta-turn (5%) were also present. The N-terminal amino acid sequence was determined for the first 18 residues. No sequence homology with other S layer proteins was found.
Dubreuil
J D
JD
Department of Biochemistry, University of Victoria, British Columbia, Canada.
Logan
S M
SM
Cubbage
S
S
Eidhin
D N
DN
McCubbin
W D
WD
Kay
C M
CM
Beveridge
T J
TJ
Ferris
F G
FG
Trust
T J
TJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification; D002168:Campylobacter fetus / Q000032:analysis / Q000276:immunology / Q000648:ultrastructure*; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005613:Freeze Etching; D005614:Freeze Fracturing; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight
D000596:Amino Acids; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D008565:Membrane Proteins