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 electrophoret...

Full description

Saved in:
Bibliographic Details
Published in:Bio/Technology 1990-11, Vol.8 (11), p.1030-1033
Main Authors: Makoff, A. J., Oxer, M. D., Ballantine, S. P., Fairweather, N. F., Charles, I. G.
Format: Article
Language:English
Subjects:
Agriculture
Amino Acid Sequence
Antigens, Bacterial - biosynthesis
Antigens, Bacterial - genetics
Antigens, Surface - biosynthesis
Antigens, Surface - genetics
Base Sequence
Bioinformatics
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Bordetella pertussis - genetics
Bordetella pertussis - immunology
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Gene Expression
Life Sciences
Molecular Sequence Data
Plasmids
Recombinant Proteins - biosynthesis
research-paper
Ribosomes - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0733-222X
1087-0156
1546-1696
DOI:10.1038/nbt1190-1030