Complex of translation elongation factors (eEF-1H): in vitro reconstitution from recombinant proteins

摘要

Translation elongation step in higher eukaryotes requires the function of three different elongation factors, eEF-1A, eEF-1B and eEF-2. Eukaryotic elongation factor 1A (eEF-1A) is a 50 kDa G-protein that binds aminoacyl-tRNAs in a GTP-dependent manner and delivers them to A site of the ribosome. Regeneration of active eEF-lA~*GTP form is assured by guanine nucleotide exchange factor eEF-1B, which contains three subunits α, β and γ. eEF-1Bα and eEF-1Bβ catalyze the GDP/GTP exchange on eEF-1A, whereas the eEF-1Bγ is considered to be a structural compo-nent. Complex of eEF-1A and three subunits of eEF-1B are called eEF-1H — the heavy form of eEF1A. Although several models of macromolecular organization of this complex have been proposed, they are contradictory to each other with respect to subunit stoichiometric ratio and molecular masse of the complex. The aim of present study was to investigate eEF-1Bαβγ complex formation from re-combinant proteins using native gel electrophoresis and gel filtration approaches. Methods. The amount of eEF- 1 H complex in eukaryotic cells is too low to be sufficient for in vitro structural studies. That was the reason to create bacterial producent strains that allowed us to obtain preparative amounts of individual recombinant subunits. eEF-1Bα and eEF-1Bβ were purified from E. coli as GST-fusion proteins with subsequent removing of GST moiety, whereas eEF-1Bγ bearing poly-histidine tag was purified by affinity chromatography on Ni-NTA matrix. The activity of eEF- iBα and eEF-1Bβ was checked by in vitro [~3H]GDP/GDP exchange assay. To visualize the complex of different recombinant subunits, we used agarose gel electrophoresis in native conditions. The molecular masses of reconstituted complexes was determined by gel filtration technique. After the gel filtration the protein composition of each peak was checked up by SDS PAGE. Results. By agarose gel in native conditions we readily detected stable eEF-1Bαγ, eEF-1Bβγand eEF-1Bαβγ complexes,when mixing equimolar amounts of each component. To determine molecular masses of the complexes, we performed gel filtration on the Superose 6 column. An analysis of individual subunits indicated unusual molecular masses of all of them, namely: eEF-1Bα was estimated to be 72 kDa, eEF-1Bβ — 350 kDa and eEF-1Bγ — 150 kDa. The theoretical molecular masses for these recombi-nant proteins were 27, 35 and 50 kDa, respectively. Gel filtration in the buffer of high ionic strength (0.5 M NaCl) containing chaotropic salt (0.5 M КSCN) did not change their elution profiles, that proves the absence of protein aggregates in the protein preparations. Mr of binary protein complexes eEF-1 Bαγ, eEF-1Bβγwas determined to be 400 and 900 kDa, respectively. Stake ternary eEF-1Bαβγ complex was also formed and according to SDS PAGE contained equimolar amounts of each subunit. Molecular masse of this complex was estimated to be more than 1 M Da. Conclusions. Individual recombinant subunits of elongation factor eEF-1 Bα, eEF-1 Bβ and eEF-1 By are able to form a stable high-molecular-masse complex in vitro. We consider that most probably eEF-1Bα and eEF1Bβ are not globular proteins, while eEF-lBγ may form a stable direr. To define precisely the com-position and molecular masse of eEF-1 H complex the equilibrium sedimentation analysis is needed.