Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm-Baculovirus Expression System.
Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm-BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm-BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Daisuke
D
Morokuma
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Jian
J
Xu
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Masato
M
Hino
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Hiroaki
H
Mon
Laboratory of Cell Migration and Signaling, Division of Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah, 23955, Saudi Arabia.
Jasmeen S
JS
Merzaban
Laboratory of DNA Replication and Recombination, Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, 4700 KAUST Thuwal, Jeddah, 23955, Saudi Arabia.
Masateru
M
Takahashi
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Takahiro
T
Kusakabe
Laboratory of Insect Genome Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan. jaemanle@agr.kyushu-u.ac.jp.
Jae Man
JM
Lee
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001665:Binding Sites; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000648:ultrastructure; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I
Baculovirus expression vector system; N-glycosylation; Silkworm; β-1, 4-Galactosyltransferase 1