Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants
Article first published online: 18 FEB 2004
The Plant Journal
Volume 38, Issue 1, pages 172–181, April 2004
How to Cite
Rohila, J. S., Chen, M., Cerny, R. and Fromm, M. E. (2004), Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. The Plant Journal, 38: 172–181. doi: 10.1111/j.1365-313X.2004.02031.x
- Issue published online: 18 FEB 2004
- Article first published online: 18 FEB 2004
- Received 20 November 2003; revised 18 December 2003; accepted 22 December 2003.
- affinity tag;
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.