Yeast strain TKY675 expresses C-terminal 6xHis-tagged elongation factor 2 (eEF2) for rapid downstream purification.
Elongation factors are proteins used in translation or protein synthesis during the process of cell cycle and elongation in some cells. In the ribosome, they facilitate translational elongation, from the formation of the first peptide bond to the formation of the last one. Specifically, elongation factor 2 catalyzes the GTP-dependent ribosomal translocation step during translation elongation. During this step, the ribosome changes from the pre-translocational (PRE) to the post-translocational (POST) state as the newly formed A-site-bound peptidyl-tRNA and P-site-bound deacylated tRNA move to the P and E sites, respectively. eEF2 catalyzes the coordinated movement of the two tRNA molecules, the mRNA and conformational changes in the ribosome.
From the laboratory of Terri Goss Kinzy, PhD, Rutgers University.
|Gene/insert name:||Expresses S. cerevisiae eEF2 containing C-terminal 6xHis-Tag|
|Strain:||Saccharomyces cerevisiae, TKY675 (Haploid)|
|Genotype:||MATa ade2 leu2 ura3 his3 leu2 trp1 eft1::HIS3 eft2::TRP1 pEFT2-6xHis LEU2 CEN|
|Format:||Spotted on filter disc|
|Storage:||Glycerol stock at -80C|
Place disc on YEPD plate, add a few drops of sterile water to the disc and streak out.
Expression and Purification of eEF2
(a) Yeast strains YEFD12h/pURA3-EFT1 (WT eEF2) and TKY675 (eEF2-6xHis) were grown overnight in YEPD to an A600 of 1.0, spotted as tenfold serial dilutions on YEPD medium and incubated for 2 d at either 303 (top panel) or 310 K (bottom panel). (b) Coomassie blue stained 10.8% SDS gel of eEF2-6xHis. Lane M, molecular-weight markers in kDa; lane 1, pool from Ni-chelating column.
Adapted from: Jørgensen, R, et al. Acta Crystallogr D Biol Crystallogr. 2002 Apr;58(Pt 4):712-5.
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