Interaction between Elongation Factors 1β and 1γ fromBombyx moriSilk Gland

Abstract

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: a (51 kDa), b (26 kDa), g (49 kDa), and d (33 kDa). The EF-1a subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1a-bound GDP is then exchanged for GTP by the EF-1 bgd complex. To facilitate analysis of the roles of the individual EF-1 b, g, and d subunits in GDP W GTP exchange on EF-1a, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1 b, EF-1g, and the carboxyl-terminal half of EF-1d were expressed, puriˆed, and examined for protein:protein interactions by gelˆltration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1 b and g subunits in a 1:1 molar ratio was detected by gelˆltration. A higher molecular weight species containing an excess of EF-1g relative to EF-1 b was also detected. The amino-terminal region of EF-1 b (amino acid residues 1-129) was su‹cient for binding to EF-1g. The carboxyl-terminal half of EF-1d did not appear to form a complex with EF-1g.

Studies on middle and posterior silk glands of silkworm (Bombyx mori) using two-dimensional electrophoresis and mass spectrometry

“…The elongation factors (EFs) (spots 38 and 47) are a group of proteins that play a regulatory role in protein biosynthesis. EF-1 from the silk gland of B. mori consists of four subunits: alpha, beta, gamma, and delta (Kamiie et al, 2003). Zhang et al identified EF-1 alpha protein in a twodimensional map of the silk gland.…”

Structural dissection of human translation elongation factor 1Bγ (eEF1Bγ): expression of full-length protein and its truncated forms

“…The large soluble aggregates of recombinant rabbit [16], B. mori [17] and multimers of the human eEF1Bg [10] were previously observed. The aggregates of rabbit eEF1Bg could be dissociated by 500 mM NaCl and at this salt concentration the molecular weight of eEF1Bg was estimated by gel-filtration to be about 140 kDa [16].…”

“…Resulted recombinant plasmids pOPINM/MBP-eEF1Bg (1-Nr) encode truncated forms of eEF1Bg in frame with MBP sequence. Constructs were prepared during the 5 th EMBO Practical Course on High throughput protein production and crystallization (16)(17)(18)(19)(20)(21)(22)(23)(24) May 2013, Harwell, United Kingdom).…”

“…The aggregates of rabbit eEF1Bg could be dissociated by 500 mM NaCl and at this salt concentration the molecular weight of eEF1Bg was estimated by gel-filtration to be about 140 kDa [16]. The preparation of the B. mori eEF1Bg was separated by gel filtration chromatography at 200 mM KCl into two fractions: large aggregates migrated close to the column void volume, whereas another fraction migrated as a single peak of about 150 kDa [17]. In contrast, in our experiments with His-eEF1Bg the high salt con- centration (up to 1 M NaCl) did not reduce the amount of large aggregates.…”

Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ fromNilaparvata lugens and its yeast-like symbiont

“…In Trypanosoma brucei, EF-1α is found in a complex that contains calmodulin, which means EF-1α may be involved in calcium-dependent signaling pathway (Kaur & Ruben, 1994). In insects, the cDNAs for EF-1α, β, γ and δ were cloned from the silk gland of Bombyx mori (Janssen & Möller, 1988;Taira et al, 1992;Kamiie et al, 1993Kamiie et al, , 2002Kamiie et al, , 2003. However, the study on its tissue distribution and expression patterns and function were very limited.…”