Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants

Jai S. Rohila, Mei Chen, Ronald Cerny, Michael E. Fromm

Research output: Contribution to journalArticle

188 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)172-181
Number of pages10
JournalPlant Journal
Volume38
Issue number1
DOIs
StatePublished - Apr 1 2004

Fingerprint

Plant Proteins
isolation techniques
Glucocorticoid Receptors
synthetic genes
nuclear localization signals
Proteins
proteins
protein isolates
calmodulin
Calmodulin-Binding Proteins
Synthetic Genes
crosslinking
green fluorescent protein
Nuclear Localization Signals
steroids
binding proteins
proteinases
transcription (genetics)
Green Fluorescent Proteins
mass spectrometry

Keywords

  • Affinity tag
  • CBP
  • Cross-linking
  • Glucocorticoid
  • NLS
  • TAP

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. / Rohila, Jai S.; Chen, Mei; Cerny, Ronald; Fromm, Michael E.

In: Plant Journal, Vol. 38, No. 1, 01.04.2004, p. 172-181.

Research output: Contribution to journalArticle

Rohila, Jai S. ; Chen, Mei ; Cerny, Ronald ; Fromm, Michael E. / Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. In: Plant Journal. 2004 ; Vol. 38, No. 1. pp. 172-181.
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