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Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants
Author(s) -
Rohila Jai S.,
Chen Mei,
Cerny Ronald,
Fromm Michael E.
Publication year - 2004
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2004.02031.x
Subject(s) - tandem affinity purification , green fluorescent protein , subcellular localization , glucocorticoid receptor , fusion protein , biology , microbiology and biotechnology , nuclear localization sequence , gene , target protein , biochemistry , affinity chromatography , chemistry , recombinant dna , enzyme
Summary A synthetic gene encoding the tandem affinity purification (TAP) tag has been constructed, and the TAP tag assayed for its effects on expression levels and subcellular localization by fusion to green fluorescent protein (GFP) as well as for its effects on steroid‐dependent translocation to the nucleus and transcription when fused to a hybrid glucocorticoid receptor. A nuclear localization signal (NLS) was detected in the calmodulin‐binding protein (CBP) domain and removed by mutation to improve the usefulness of the TAP tag. Additionally, purification improvements were made, including inhibition of a co‐purifying protease, and adding a protein cross‐linking step to increase the recovery of interacting proteins. The improved synthetic TAP tag gene and methods were used to isolate proteins interacting with the hybrid glucocorticoid receptor and to identify them by mass spectrometry. The two proteins identified, HSP70 and HSP90, are known to interact with the glucocorticoid receptor in vivo in mammalian cells and in vitro in plants.