title: Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures.
abstract: Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Microbiology, Gesellschaft für Biotechnologische Forschung, Braunschweig, FRG.
authors.forename: M J
authors.initials: MJ
authors.lastname: Walker
authors.forename: M
authors.initials: M
authors.lastname: Rohde
authors.forename: R M
authors.initials: RM
authors.lastname: Brownlie
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D010861:Fimbriae, Bacterial / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins