title: Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells.
abstract: Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78kDa which was much smaller than that (∼97kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h90K was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (∼79%) N-glycans.
authors.affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
authors.forename: Kyoung Ro
authors.initials: KR
authors.lastname: Kim
authors.forename: Yeon Kyu
authors.initials: YK
authors.lastname: Kim
authors.forename: Hogyun
authors.initials: H
authors.lastname: Cheong
authors.forename: Jaoon Y H
authors.initials: JY
authors.lastname: Kim
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D001709:Biotechnology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004331:Drosophila melanogaster; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016326:Extracellular Matrix Proteins
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C081856:LGALS3BP protein, human; D016326:Extracellular Matrix Proteins
keywords: Drosophila S2 cell; Glycoprotein; Mac-2-binding protein; N-Glycosylation; h90K