Article

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

Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX 78712, USA.
Protein Expression and Purification (impact factor: 1.59). 05/2011; 79(2):237-44. DOI:10.1016/j.pep.2011.05.005 pp.237-44
Source: PubMed

ABSTRACT 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-His(6)-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.

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Keywords

biochemical analysis
 
Escherichia coli strain Rosetta-gami 2(DE3)
 
eukaryotic elongation factor 2
 
eukaryotic elongation factor 2 kinase
 
expressed kinase
 
form suitable
 
gel filtration columns
 
kinase activity
 
mammalian enzyme
 
MAPK pathways
 
mTOR
 
phosphorylate wheat germ eEF-2
 
pivotal role
 
protein synthesis
 
recombinant human eEF-2K
 
recombinant kinase
 
Successive chromatography steps utilizing Ni-NTA affinity
 
translation regulation
 
tumor survival
 
vitro kinetic analysis