A novel strategy for the immunological tagging of cDNA constructs.
We describe the construction of pBact-myc, an expression vector that incorporates an immunological 'tag' into the produced polypeptide. When transfected into recipient cell lines, tagged protein fragments derived from any source can be visualised using a single monoclonal antibody (mAb). The neuronal-associated protein 2c (MAP2c) was tagged with a sequence encoding a peptide from the human c-myc gene. The preservation of normal function of the tagged protein was shown by transfecting it into cultured cell lines. No difference in binding ability to cellular microtubules could be observed between the myc-tagged MAP2c and the wild-type forms, and both produced the same characteristic changes in microtubule organisation. This approach is being used to study the biological function of selected fragments of MAP2c and other MAP-encoding genes. The pBact-myc expression vector represents a fast and convenient way to produce tagged polypeptides of selected sequences encoding whole proteins or fragments, for the analysis of their function in living cells.
Cravchik
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Friedrich Miescher-Institut, Basel, Switzerland.
Matus
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D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D000939:Epitopes; D016259:Genes, myc; D005822:Genetic Vectors; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016324:Sequence Tagged Sites
D018076:DNA, Complementary; D000939:Epitopes; D008869:Microtubule-Associated Proteins