Expression and secretion of the S2 subunit of pertussis toxin in Bacillus brevis

We have been trying to develop a mass production system for each of the subunits (S2, S3, S4, S5) of the pertussis toxin B oligomer (PTB) by using a Bacillus brevis-pNU212 system. In consequence a moderately efficient expression-secretion system for S2 was constructed by fusing the mature S2 gene from Bordetella pertussis Tohama with the signal-peptide coding region of pNU212 and by introducing the plasmid pNU212-S2 into B. brevis HPD31 by electroporation. The clone producing S2 secreted about 70 mg of recombinant S2 (rS2) per liter of PY-erythromycin medium after 5 days incubation at 37 degrees C. The rS2 purified by an ammonium sulfate fractionation at 30-50% saturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was identical to the native S2 in respect to the molecular weight determined by SDS-PAGE and the amino terminal amino acid sequence. The nucleotide sequence of the S2 gene in B. pertussis Tohama inserted into pNU212 was identical with that of the S2 gene in other virulent B. pertussis strains except that an adenine at position 52 of the latter was replaced by a guanine in the former, causing an amino acid substitution (glycine in the former for serine in the latter) at position 18.