Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins.
We describe the construction of pBact-NGFP and pBact-CGFP, two expression vectors that incorporate green fluorescent protein (GFP) as a fluorescent tag at the N- or C terminus of the produced protein. When transfected into recipient cells, GFP-tagged proteins can be visualised in the living cells using standard fluorescence microscopy techniques. Using these expression vectors, we have produced GFP-tagged versions of the neuronal microtubule-associated proteins (MAP), MAP2c and Tau34, in a number of different cell types. Both GFP-MAP2c and GFP-Tau34 were fluorescent and retained their ability to bind to microtubules. The pBact-NGFP and pBact-CGFP expression vectors represent a fast and convenient way to produce fluorescently tagged polypeptides of selected sequences encoding whole proteins or fragments for the analysis of function and dynamic events in living cells.
Ludin
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B
Friedrich Miescher Institut, Basel, Switzerland.
Doll
T
T
Meili
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R
Kaech
S
S
Matus
A
A
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D037401:Scyphozoa; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins