title: Expression and secretion of pertussis toxin subunit S1 in Bacillus subtilis.
abstract: Pertussis toxin (PT) is an important virulence determinant of Bordetella pertussis and one of the major protective antigens against whooping cough. The genes coding for PT have recently been cloned, but attempts to express them in Escherichia coli have been unsuccessful. We therefore explored the possibility of expressing these genes in Bacilius subtilis for which efficient vectors are available. The lack of endotoxin in the Gram-positive Bacillus might be an additional advantage for the production of a vaccine component. A DNA fragment coding for S1, one of the subunits of pertussis toxin, was inserted into an alpha-amylase secretion vector and the recombinant plasmid was introduced into B. subtilis. This resulted in high expression of S1, most of which was secreted and therefore found in the culture supernatant. This supernatant had ADP-ribosylating activity similar to that of PT. Western blot with antiserum to B. pertussis holotoxin showed several proteins ranging in size from 28 kDa to 20 kDa reacting in specific manner. About 10% of the protein recognized by the antiserum was of the size expected for native-size S1. The total amount of S1 proteins (full size and truncated) in the culture supernatant was about 100 mg/l. S1 protein made in B. subtilis was partially purified using chromatography with P-cellulose and Blue Sepharose. This preparation was used to immunize rabbits; the immune serum thus obtained recognized subunit S1 of native pertussis toxin.
authors.affiliation: National Public Health Institute, Helsinki, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Runeberg-Nyman
authors.forename: O
authors.initials: O
authors.lastname: Engström
authors.forename: S
authors.initials: S
authors.lastname: Löfdahl
authors.forename: S
authors.initials: S
authors.lastname: Ylöstalo
authors.forename: M
authors.initials: M
authors.lastname: Sarvas
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D001886:Bordetella pertussis / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015247:Deoxyribonuclease HindIII; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D015183:Restriction Mapping; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D015247:Deoxyribonuclease HindIII