Cloning and expression in Escherichia coli of the Ibc protein genes of group B streptococci: Binding of human immunoglobulin A to the beta antigen

Abstract

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 λL47.1 in Escherichia coli K-12. Recombinant phage plaques in the bank were immunoblotted by using anti-α- and anti-β-specific antibodies directed towards the Ibc proteins purified from the streptococcal cell surface, and hybrid phages expressing the α protein (λα+) and the β protein (λβ+) were identified. DNA inserts in these phages were subcloned into E. coli high-copy-number plasmid vectors to produce stable α+ (pPHC10) and β+ (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 α protein, previously undetected, was identified; this species may be the native α 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 β protein, but not the α protein, bound human immunoglobulin A (IgA) in Western blots, and neither protein bound IgG or IgM.