Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants.
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.
Rohila
Jai S
JS
Plant Science Initiative, University of Nebraska, Lincoln, NE 68588, USA.
Chen
Mei
M
Cerny
Ronald
R
Fromm
Michael E
ME
D000345:Affinity Labels; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012231:Rhizobium / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D019913:Nuclear Localization Signals; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins