A rapid method of epitope mapping

Abstract

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Ba131 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action.This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101 -110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1 -60).The 79-kDa form ('subunit A') of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101 -110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.The precise mapping of epitopes recognized by monoclonal antibodies is important for the definition of immunogenic [l] In most cases in which monoclonal antibodies are available for a protein the corresponding cDNA has been cloned or can easily be cloned. We here describe a technique involving acellular transcription/translation of the cloned cDNA, in which use is made of spontaneously occurring or provoked premature terminations of translation. Immunoprecipitation of the C-terminally truncated proteins allows, in a few rapid experiments, the precise mapping of corresponding epitopes. This method has been applied using a series of monoclonal antibodies raised against the rabbit progesterone receptor. It has led to the definition of the immunogenic sites and to the cartography of the various forms of this regulatory protein. Special emphasis has been put on the study of the so-called 'A form' of the receptor (molecular mass 79 kDa) which is considered by some to be a distinct physiological entity in chick [15] and human [16]. However, our studies performed in the rabbit suggest that it arises by artefactural proteolysis 1171 of the native receptor ('B form', apparent molecular mass in SDS/polyacrylamide gels: 110 kDa, molecular mass deduced from sequence: 98554 Da 1181)....

Specific binding of progesterone receptor to progesterone‐responsive elements does not require prior dimerization

“…The Let 126 antibody was then used. Let 126 binds only to the 110-kDa progesterone receptor (B form), but not to the 79-kDa (A form) species [13]. Incubation with this antibody shifted the position of both complexes, thus showing that both contained the N-terminal region of the receptors.…”

“…The Centricon-30 system was used to reduce the ionic strength of the supernatant to 20 mM KCl and to concentrate the extract. The monoclonal antibody for the progesterone receptor, Let 126 [13], was added to the supernatant at a final concentration of 300 pg/ ml and incubated for 2 h at 4"C, with gentle agitation. The resulting comulexes were then incubated with an 80-fold expared in 10 mM Tris, pH 7 [14].…”

“…The resulting comulexes were then incubated with an 80-fold expared in 10 mM Tris, pH 7 [14]. The pellets were solubilized in Laemmli buffer (62.6mM Tris, pH6.8, 2% SDS, 5% 2-mercaptoethanol) [15], before analysis by Western blotting, using the monoclonal antibody Mi60 specific for the progesterone receptor [13].…”

Sumoylation of the Progesterone Receptor and of the Steroid Receptor Coactivator SRC-1

“…After a final wash with PBS, the bound proteins were eluted by boiling in Laemmli loading buffer and subjected to a 6.4% SDS-PAGE. The proteins were analyzed by Western blotting using primary anti-PR monoclonal antibody Let126 (45) or anti-HA 12CA5 monoclonal antibody (Roche Molecular Biochemicals) at a concentration of 2 g/ml. The ECL system (Amersham Biosciences) was used for band detection.…”

The tumor suppressor activity induced by adenovirus-mediated BRCA1 overexpression is not restricted to breast cancers

“…59 The secondary peroxidase-conjugated goat anti-mouse IgG was from Jackson Immunoresearch Laboratories (West grove, PA, USA), the donkey anti-rabbit IgG (Na934) was from Amersham Life Sciences (Buckin- …”