S. Rao et Jw. Bodley, EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE G-DOMAIN OF SACCHAROMYCES-CEREVISIAE ELONGATION-FACTOR-2, Protein expression and purification, 8(1), 1996, pp. 91-96
Protein synthesis elongation factor 2 (EF-2), a member of the G protei
n superfamily, catalyzes the movement of the ribosome along mRNA in a
reaction driven by the hydrolysis of GTP. In the present study, we hav
e expressed, purified, and characterized the G domain of Saccharomyces
cerevisiae EF-2 to define its functional properties. A peptide of 185
amino acids was found to be sufficient to bind guanine nucleotides wh
en expressed in Escherichia coli either as a fusion with the maltose b
inding protein or as a truncation. The fusion was expressed as a solub
le, active protein, while the truncation was expressed as an insoluble
protein that required renaturation in order to function, Similar to n
ative EF-2, both expression products bound GDP, but they did so with s
ignificantly lower affinity (K-d ca. 200 mu M for both the fusion and
the truncation vs ca. 0.5 mu M for native EF-2). However, in contrast
to native EF-2 which requires the ribosome for GTP hydrolysis, the iso
lated G domain hydrolyzed GTP (K-m ca. 40 mu M; turnover rate ca. 0.5
min(-1)) in the absence of the ribosome. We conclude that the G domain
residues necessary for GTP binding and hydrolysis are located in the
first 185 amino acids of S. cerevisiae EF-2. (C) 1996 Academic Press,
Inc.