Protective surface antigen P69 of Bordetella pertussis: its characterization and very high level expression in Escherichia coli.
The surface antigen, P69 of Bordetella pertussis, an N-terminal fragment of the precursor protein, P93, is likely to be an important component of future subunit vaccines against whooping cough. We have expressed several defined N-terminal fragments of P93 in E. coli and compared their electrophoretic mobilities with that of purified P69 from B. pertussis. These experiments show that P69 is considerably smaller than the 69 kD originally estimated from its gel mobility and is probably 60.4 kD in size. Our initial plasmids expressed only very low levels of this antigen. We diagnosed the limiting factor to be a poor ribosome binding site (RBS) by demonstrating a large stimulation of expression on a two-cistron plasmid. The limitation of expression could be completely overcome by only two base changes close to the initiation codon, resulting in a further increase in expression of P69 at levels to 30-40% total cell protein. Although the protein accumulated as insoluble inclusion bodies, it could be solubilized by guanidinium chloride.
Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, U.K.
A J
AJ
Makoff
M D
MD
Oxer
S P
SP
Ballantine
N F
NF
Fairweather
I G
IG
Charles
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics*; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism
D000942:Antigens, Bacterial; D000954:Antigens, Surface; D011994:Recombinant Proteins