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 th e subunits (S2, S3, S4, S5) of the pertussis toxin B oligomer (PTB) by using a Bacillus brevis-pNU212 system. In consequence a moderately ef ficient expression-secretion system for S2 was constructed by fusing t he mature S2 gene from Bordetella pertussis Tohama with the signal-pep tide coding region of pNU212 and by introducing the plasmid pNU212-S2 into B. brevis HPD31 by electroporation. The clone producing S2 secret ed about 70 mg of recombinant S2 (rS2) per liter of PY-erythromycin me dium after 5 days incubation at 37 degrees C. The rS2 purified by an a mmonium 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-PA GE and the amino terminal amino acid sequence. The nucleotide sequenc e of the S2 gene in B. pertussis Tohama inserted into pNU212 was ident ical with that of the S2 gene in other virulent B. pertussis strains e xcept that an adenine at position 52 of the latter was replaced by a g uanine in the former, causing an amino acid substitution (glycine in t he former for serine in the latter) at position 18. Copyright (C) 1996 Elsevier Science Ltd.