Purification and characterization of S layer proteins from Clostridium difficile GAI 0714.
The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.
Takeoka
A
A
Department of Food Microbiology, Tokushima University School of Medicine, Japan.
Takumi
K
K
Koga
T
T
Kawata
T
T
D000596:Amino Acids / Q000032:analysis; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry; D002473:Cell Wall / Q000737:chemistry; D016360:Clostridioides difficile / Q000032:analysis* / Q000276:immunology / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007526:Isoelectric Point; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D010449:Peptide Mapping
D000596:Amino Acids; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins