Abstract
Fusion proteins are used to define with precision an evolutionarily conserved domain on the carboxyl-terminal portion of the chromoprotein phytochrome. Simultaneously, assignments of two other epitopes are made with significantly greater precision, while the location of a fourth is confirmed. The epitope-mapping method that is described here is systematic, using complementary, overlapping nested sets of fusion proteins of predefined sequence rather than randomly generated peptides. Moreover, in contrast to previous methods, this approach yields rigorous and unambiguous assignments because it relies solely upon the ability of an antibody to detect a given polypeptide. A cDNA fragment encoding phytochrome amino acids 464-1129, which is its carboxyl terminus, was identified in lambda gt11 and subcloned in frame into the lacZ alpha sequence of pUC18. Four nested sets of subclones in pUC18 were created by digestion with selected restriction endonucleases and with the exonuclease Bal31. Fusion proteins were analyzed by immunoblotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The epitope for monoclonal antibody Oat-13 was confirmed to be between residues 551 and 617, while the epitopes for Oat-8 and Oat-28 were narrowed to 624-686 and 624-747, respectively. The epitope recognized by Pea-25, Pea-2, and Oat-15 was resolved unequivocally to a sequence of only seven residues (residues 765-771): N-Pro-Ile-Phe-Gly-Ala-Asp-Glu-C.
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Claims 5
Claim · model-written summary
Fusion protein sets provide systematic and unambiguous epitope assignments.
Verbatim
e0“in contrast to previous methods, this approach yields rigorous and unambiguous assignments because it relies solely upon the ability of an antibody to detect a given polypeptide”
Claim · model-written summary
The Oat-13 epitope was confirmed between residues 551 and 617.
Not reportedTaxon
Verbatim
e0“The epitope for monoclonal antibody Oat-13 was confirmed to be between residues 551 and 617”
Claim · model-written summary
The Oat-8 epitope was narrowed to 624-686.
Not reportedTaxon
Verbatim
e0“the epitopes for Oat-8 and Oat-28 were narrowed to 624-686 and 624-747, respectively”
Claim · model-written summary
The Oat-28 epitope was narrowed to 624-747.
Not reportedTaxon
Verbatim
e0“the epitopes for Oat-8 and Oat-28 were narrowed to 624-686 and 624-747, respectively”
Claim · model-written summary
Pea-25, Pea-2 and Oat-15 recognized a seven-residue sequence.
Not reportedTaxon
Verbatim
e0“The epitope recognized by Pea-25, Pea-2, and Oat-15 was resolved unequivocally to a sequence of only seven residues (residues 765-771): N-Pro-Ile-Phe-Gly-Ala-Asp-Glu-C”