Cloning and expression of Bombyx mori silk gland elongation factor 1gamma in Escherichia coli.
Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of alpha-, beta-, gamma-, and delta-subunits. EF-1alpha GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1betagammadelta catalyzes the exchange of EF-1alpha-bound GDP for exogenous GTP and stimulates the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosomes. EF-1gamma cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1gamma shares 67.3% amino acid identity with Artemia salina EF-lgamma. The N-terminal domain (amino acid residues 1-211) of silk gland EF-lgamma is 29.3% identical to maize glutathione S-transferase. We demonstrated that silk gland EF-lgamma bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1gamma may have the capacity to bind to glutathione.
Kamiie
Katsuyoshi
K
Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University, Japan. kamiie@aomori-u.ac.jp
Nomura
Yoshitaka
Y
Kobayashi
Satoru
S
Taira
Hideharu
H
Kobayashi
Kohmei
K
Yamashita
Tetsuro
T
Kidou
Shin-ichiro
S
Ejiri
Shin-ichiro
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005088:Exocrine Glands / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1 / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D020648:Peptide Elongation Factor 1; D005982:Glutathione Transferase; D005978:Glutathione