title: High-level expression of human glycosyltransferases in insect cells as biochemically active form.
abstract: cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.
authors.affiliation: Department of Life Science, Sogang University, Mapo-gu, Seoul 121-742, Republic of Korea.
authors.forename: Hyung Gu
authors.initials: HG
authors.lastname: Kim
authors.forename: Sung Mo
authors.initials: SM
authors.lastname: Yang
authors.forename: Young Choon
authors.initials: YC
authors.lastname: Lee
authors.forename: Su Il
authors.initials: SI
authors.lastname: Do
authors.forename: In Sik
authors.initials: IS
authors.lastname: Chung
authors.forename: Jai Myung
authors.initials: JM
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007075:Immunoglobulin M / Q000737:chemistry; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000737:chemistry; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D007075:Immunoglobulin M; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D016695:Glycosyltransferases; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase