Epitope mapping of recombinant antigens by transposon mutagenesis.
We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.
Morris
G E
GE
MRIC Biotechnology Group, NE Wales Institute, Deeside Clwyd, UK.
Nguyen thi Man
Sedgwick
S G
SG
D000911:Antibodies, Monoclonal; D000941:Antigens / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D004251:DNA Transposable Elements; D016189:Dystrophin / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry*; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D000941:Antigens; D004251:DNA Transposable Elements; D016189:Dystrophin; D011994:Recombinant Proteins