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Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
17302 42658 2013 4 PDF Available
Title
Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells
Abstract

•Production of human 90K (h90K) as a potential therapeutic glycoprotein in insect Drosophila S2 cell expression system.•Efficient secretion of recombinant h90K into culture medium in S2 cells.•S2-derived recombinant h90K has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major N-glycans.

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 28 mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78 kDa which was much smaller than that (∼97 kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60 kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18 kDa) 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.

Keywords
h90K; Mac-2-binding protein; Glycoprotein; N-Glycosylation; Drosophila S2 cell
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Expression and N-glycan analysis of human 90K glycoprotein in Drosophila S2 cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issue 3, 15 August 2013, Pages 170–173
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us