Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli

Olga Abramczyk, Clint D.J. Tavares, Ashwini K. Devkota, Alexey G. Ryazanov, Benjamin E. Turk, Austen F. Riggs, Bulent Ozpolat, Kevin N. Dalby

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function, and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange, and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ∼85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme.

Original languageEnglish (US)
Pages (from-to)237-244
Number of pages8
JournalProtein Expression and Purification
Issue number2
StatePublished - Oct 2011


  • Calmodulin
  • Elongation factor 2 kinase
  • eEF-2K

ASJC Scopus subject areas

  • Biotechnology


Dive into the research topics of 'Purification and characterization of tagless recombinant human elongation factor 2 kinase (eEF-2K) expressed in Escherichia coli'. Together they form a unique fingerprint.

Cite this