Molecular and immunochemical characterization of recombinant Escherichia coli containing the spaA gene region of Streptococcus sobrinus.
We identified and characterized a recombinant Escherichia coli containing the entire gene for surface protein antigen A (spaA) of Streptococcus sobrinus 6715. The recombinant E. coli was isolated from a cosmid gene bank of size-fractionated S. sobrinus DNA fragments, and recombinants expressing the SpaA protein were detected immunologically. Subcloning experiments showed that the DNA sequences encoding the SpaA protein could be isolated on two contiguous EcoRI fragments, 3.7 and 3.3 kilobases (kb) in size, both contained on a 16.2-kb BglII fragment. Southern blot hybridization experiments using the EcoRI fragments to probe genomic DNAs from various serotypes of the mutans group of streptococci revealed DNA sequence homology not only to S. sobrinus 6715 (serotype g) chromosomal DNA but also to S. sobrinus serotype d DNA. Weak hybridization signals to Streptococcus mutans serotypes c, e, and f and to Streptococcus cricetus serotype a were observed with the 3.3-kb EcoRI fragment. These results suggest that the coding sequence for the spaA gene is probably conserved in S. sobrinus strains. Plasmid-encoded polypeptides made in E. coli minicells revealed that transcription of the spaA gene was initiated on the 3.7-kb EcoRI fragment and that its product size was about 210 kilodaltons. The cloned SpaA protein was purified from the periplasmic protein of E. coli, and monospecific antiserum against the cloned product was prepared in rabbits. The data obtained from various physiochemical and immunological procedures allowed us to conclude that the coding sequence for the entire spaA gene of S. sobrinus 6715 had been successfully cloned in E. coli and that faithful expression of the cloned product could be obtained.
Ogundipe
J O
JO
Department of Microbiology, Meharry Medical College, Nashville, Tennessee 37208.
Holt
R G
RG
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005779:Immunodiffusion; D013291:Streptococcus / Q000235:genetics*; D013295:Streptococcus mutans / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant