Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris.
cDNAs encoding three alpha-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (38-44% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.
Nishimoto
Mamoru
M
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Mori
Haruhide
H
Moteki
Tsuneharu
T
Takamura
Yukiko
Y
Iwai
Gaku
G
Miyaguchi
Yu
Y
Okuyama
Masayuki
M
Wongchawalit
Jintanart
J
Surarit
Rudee
R
Svasti
Jisnuson
J
Kimura
Atsuo
A
Chiba
Seiya
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001516:Bees / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D010843:Pichia; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D000520:alpha-Glucosidases / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000520:alpha-Glucosidases