Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: binding of human immunoglobulin A to the beta antigen.
Total-cell DNA isolated from a highly virulent serotype Ic strain of a group B streptococcus was used to construct a gene bank with bacteriophage lambda L47.1 in Escherichia coli K-12. Recombinant phage plaques in the bank were immunoblotted by using anti-alpha- and anti-beta-specific antibodies directed towards the Ibc proteins purified from the streptococcal cell surface, and hybrid phages expressing the alpha protein (lambda alpha +) and the beta protein (lambda beta +) were identified. DNA inserts in these phages were subcloned into E. coli high-copy-number plasmid vectors to produce stable alpha + (pPHC10) and beta + (pPHC8 and pPHC33) recombinant plasmids, and restriction maps of the cloned streptococcal sequences were constructed. Antibodies against the two Streptococcus-derived proteins reacted with high-molecular-weight polypeptides made in E. coli cells carrying the corresponding hybrid plasmids and with several degradation peptides from them. A 190-kilodalton alpha protein, previously undetected, was identified; this species may be the native alpha protein or a precursor of it. In addition, mutagenesis of the cloned sequences was carried out by using the omega fragment to determine the direction of transcription. In E. coli, the beta protein, but not the alpha protein, bound human immunoglobulin A (IgA) in Western blots, and neither protein bound IgG or IgM.
D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007070:Immunoglobulin A / Q000378:metabolism*; D066298:In Vitro Techniques; D009154:Mutation; D011485:Protein Binding; D013292:Streptococcus agalactiae / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C039214:Ibc proteins, Streptococcus; D007070:Immunoglobulin A