Ks. Hong et al., PURIFICATION AND CHARACTERIZATION OF M3 PROTEIN EXPRESSED ON THE SURFACE OF GROUP-A STREPTOCOCCAL TYPE-3 STRAIN C203, FEMS immunology and medical microbiology, 12(1), 1995, pp. 73-82
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.