RRC ID 50440
Author Morokuma D, Xu J, Hino M, Mon H, Merzaban JS, Takahashi M, Kusakabe T, Lee JM.
Title Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm-Baculovirus Expression System.
Journal Mol. Biotechnol.
Abstract 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.
Volume 59(4-5)
Pages 151-158
Published 2017-5
DOI 10.1007/s12033-017-0003-1
PII 10.1007/s12033-017-0003-1
PMID 28342150
MeSH Animals Baculoviridae / genetics* Binding Sites Bombyx / genetics Bombyx / metabolism* Cloning, Molecular / methods Enzyme Activation Enzyme Stability Galactosyltransferases / biosynthesis* Galactosyltransferases / chemistry* Galactosyltransferases / ultrastructure Humans Protein Binding Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism Transfection / methods*
IF 1.815
Resource
Silkworms n17