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

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.

Figure 1. Schematic representation of cloning and expression strategy for eEF-2K

(A) Strategy for eEF-2K cloning into the pET-32a vector, including the replacement of EK (Enterokinase) with TEV (Tobacco Etch Virus) protease cleavage site. (B) Schematic representation of the expression product of eEF-2K from the pET-32a vector. (C) Primary amino acid sequence of the tagless recombinant kinase, (G-D-I)-eEF-2K. Composition of purified eEF-2K was analyzed for consistency with the known primary amino acid sequence by amino acid analysis as described under ‘Materials and Methods’.

Figure 2. Purification of eEF-2K

(A) Gel filtration chromatography on a HiPrep™ 26/60 Sephacryl S-200 column. Ni-NTA eluate was concentrated and applied to the gel filtration column. Peak (a) contains aggregated eEF-2K while peak (b) contains monomeric eEF-2K. (B) Ion exchange chromatography on a Mono Q HR 10/10 anion exchange column. Peak (b) fractions from the previous purification step were pooled and then incubated with TEV Protease, resulting in cleavage of the Trx-His-tag. Tagless-eEF-2K was applied to the Mono Q 10/10 column. (C) Gel filtration chromatography on a HiLoad™ 16/60 Superdex 200 column. Peak (a) fractions from the Mono Q column were pooled and applied to the gel filtration column. (D) Samples from the various purification steps were resolved by SDS-PAGE: BenchMark™ protein ladder (lane 1); Bacterial lysate (lane 2); Ni-NTA agarose affinity chromatography (lane 3); 26/60 Sephacryl S-200 gel filtration chromatography (lane 4); Mono Q 10/10 anion exchange chromatography following cleavage of Trx-His-tag by TEV Protease (lane 5); HiLoad™ Superdex 200 gel filtration chromatography (lane 6).

Figure 3. Light scattering analysis of unphosphorylated eEF-2K

The continuous patterns represent the refractive index signal for duplicate runs; horizontal lines represent the calculated molar mass. Analysis indicates that eEF-2K is monomeric with mass of ~85 kDa. The protein concentration at the maximum of the upper curve is ~1.6 μM reflecting a ~30-fold dilution of the injected sample.

Figure 4. Analysis of the kinase activity of eEF-2K

Wheat germ eEF-2 dependence assays were performed using 2 nM eEF-2K and 0-20 μM eEF-2 in a suitable buffer as described under ‘Materials and Methods’. The data were fitted to equation 1, where $K_{\mathrm{M}}^{\mathrm{app}}=5.9\pm 0.4\mathrm{\mu M}$ and $V_{\max }^{\mathrm{app}}=0.04\pm 0.001\mathrm{\mu M}.{\mathrm{s}}^{­1}$. Kinase activity was determined by measuring the rate of phosphorylation of eEF-2 (μM.s^-1)