Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis.
Monoclonal IgGs were shown to be useful for the specific inhibition of a set of activities of the recA protein, a key protein in homologous genetic recombination. The mapping of the epitopes for these IgGs and site-directed mutagenesis based on the mapping will facilitate location of the functionally active sites on the tertiary structure of the protein, which is being solved by means of physicochemical techniques. We developed a novel technique for region-specified mutagenesis and applied the technique to epitope mapping. Using the polymerase chain reaction in the presence of deoxyinosine triphosphate, we introduced random base substitutions specifically into a region of the recA gene defined by a pair of primers. RecA mutants exhibiting altered antigenicity were selected, in plaque-immunoblotting experiments, from libraries of mutagenized recA genes constructed on the lambda gt11 expression vector. Mutant recA genes were obtained at the frequency of about 10(-2) among the plaques expressing fused recA genes and then each one was expressed as a whole protein, which was characterized by enzyme-linked immunosorbent assay. Analyzing the DNA sequences of the mutant recA genes, we located at the amino acid sequence level the epitopes for two anti-recA IgGs which could not be located in previous studies. One of the antibodies was shown to prevent self-assembly of the recA protein and the other was suggested to inhibit the binding of double-stranded DNA. Thus, the active sites involved in these functions would be located in the space around or near the relevant epitope.
Laboratory of Microbiology, RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan.
M
M
Ikeda
K
K
Hamano
T
T
Shibata
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003062:Codon; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007074:Immunoglobulin G; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D011938:Rec A Recombinases / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D003062:Codon; D000939:Epitopes; D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D011938:Rec A Recombinases