A recombinant phage that produced a polypeptide possessing the characteristics of pneumolysin, the membrane damaging toxin of the pneumococcus, was isolated from a bank of pneumococcal sequences in ?gt10. Subclones carrying the pneumolysin gene in various plasmids were haemolytic regardless of the orientation of the insert. The nucleotide sequence of a 5 kb fragment carrying the pneumolysin gene was determined. An open reading frame 1413 bp long was identified that when translated encoded a polypeptide with 471 amino acids and a molecular weight 52.8 kD. The N-terminal amino acid sequence of the predicted protein was identical to that of native pneumolysin. A single cysteine residue was present at position 428 in the amino acid sequence. Comparison of the DNA and amino acid sequences of pneumolysin with streptolysin O (SLO) revealed extensive homology in the amino acid sequence. The longest region of identity was a sequence of 12 amino acids surrounding the unique cysteine. A hybrid gene consisting of the 5' region of the pneumolysin gene and the 3' end of the SLO gene was constructed. The fusion polypeptide was made in E. coli, but possessed a very low haemolytic activity. Using the technique of oligonucleotide-mediated site-directed mutagenesis, two mutant genes were constructed in which the cysteine codon was changed to either a glycine or serine codon. Modified toxins when purified from E. coli had a specific activity of about 1-2 % that of wild type pneumolysin.

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