PURIFICATION AND CHARACTERIZATION OF M3 PROTEIN EXPRESSED ON THE SURFACE OF GROUP-A STREPTOCOCCAL TYPE-3 STRAIN C203

Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C m ice with whole M(+) bacteria in incomplete Freund adjuvant and the res ulting mAbs for M3 protein have been selected by an indirect immune-fl uorescent technique using formaldehyde-fixed M(+) and M(-) bacteria. F our mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M(+) bacteria after treatment with N-acetyl muramidase a nd lysozyme. The purified 65 kDa protein neutralized the phagocytic ac tivity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The eviden ce indicates that the 65 kDa protein is M3 protein. The M3 protein bou nd not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromat ography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the inta ct molecule appeared. N-terminal amino acid sequence analysis showed t hat 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, be ing identical at positions 1-5 and 198-202 to the M3 gene derived prot ein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed t o be cleavage products, may be derived from the C-terminal part and N- terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human bloo d in the presence of M(-) bacteria was investigated, the M3 protein wa s responsible for the organism's resistance to attack by phagocytic ce lls.