THE S-LAYER COMPOSED OF 2 DIFFERENT PROTEIN SUBUNITS FROM CLOSTRIDIUM-DIFFICILE GAI-1152 - A SIMPLE PURIFICATION METHOD AND CHARACTERIZATION

Surface protein layer (S layer) of Clostridium difficile GAI 1152 was a tetragonal array with each side of 7.8 nm and interior angle of 77.8-degrees, which was composed of the two protein subunits with respective molecular weights of 38 kDa and 42 kDa. The S layer protein subunits were directly isolated from the whole cells by simple procedures involving extraction of the whole cells with 6 M GHCl and precipitation by 50% saturation with ammonium sulfate and purified by using DEAE-Sepharose CL-6 B column chromatography followed by DEAE-5 PW equipped to HPLC. Amino acid analysis showed that both the subunits were acidic polypeptide and neither proline nor cysteine were present. The 38 kDa subunit exhibited a single isoelectric form (pI 4.0), whereas the 42 kDa subunit showed multiple isoelectric forms ranging from 5.5 to 6.3 of pI. Limited proteolysis of the both subunits with Staphylococcus aureus V8 enzyme showed quite dissimilar peptide maps between them, suggesting marked differences in primary structures.