High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics

J Struct Funct Genomics. 2005;6(2-3):143-7. doi: 10.1007/s10969-005-1908-7.


The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis Proteins / isolation & purification*
  • Carbon Isotopes / metabolism
  • Chromatography, Affinity
  • Escherichia coli
  • Nitrogen Isotopes / metabolism
  • Proteomics / methods*
  • Quality Control
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism
  • Selenomethionine / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • Arabidopsis Proteins
  • Carbon Isotopes
  • Nitrogen Isotopes
  • Recombinant Proteins
  • Selenomethionine