In vitro gene expression for the localization of antigenic determinants: application to the E. coli tryptophan synthase beta 2 subunit.
Gene expression of the beta subunit of E. coli tryptophan synthase in an E. coli cell-free transcription-translation system proceeds by pauses and produces a discrete but quite continuous pattern of nascent chains starting from the N terminus and ranging in size up to the 44 kDa end product corresponding to the completed beta chains. Using specific immunoadsorption of [35S]Met radiolabelled nascent chains by different monoclonal antibodies directed against the beta 2 subunit of E. coli tryptophan synthase, the size of the smallest N-terminal fragment reacting with each antibody has been determined by SDS electrophoretic analysis of the immunoadsorbed polypeptides. The immunoadsorption assay is performed in solution under conditions avoiding the usual drawbacks of solid phase immunoassay. This approach, in combination with the results obtained with a DNA fragment library permitted us to localize the antigenic determinants recognized by the monoclonal antibodies. The proposed method could help to localize rapidly the C-terminal boundary of an epitope, before starting systematic and precise mapping by other approaches. Moreover, the method described may be of general interest for the rapid production of a large set of C-terminal truncated polypeptides for studies of antigen-antibody recognition.
Friguet
B
B
Unité de Biochimie Cellulaire (C.N.R.S. U.R.A. 1129), Institut Pasteur, Paris, France.
Fedorov
A N
AN
Djavadi-Ohaniance
L
L
D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002474:Cell-Free System; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010449:Peptide Mapping / Q000379:methods*; D014176:Protein Biosynthesis; D015203:Reproducibility of Results; D014158:Transcription, Genetic; D014367:Tryptophan Synthase / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D014367:Tryptophan Synthase