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Expression of functional human transferrin in stably transfected Drosophila S2 cells

Title
Expression of functional human transferrin in stably transfected Drosophila S2 cells
Authors
Lim, HJKim Yeon KyuHwang, DSCha Hyung Joon
POSTECH Authors
Hwang, DS
Date Issued
Aug-2004
Publisher
wiley
Abstract
Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His(6)) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 mug/mL, producing 6.8 mug/mL/ day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His(6)-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.
Keywords
HUMAN SERUM TRANSFERRIN; RECOMBINANT HUMAN TRANSFERRIN; GREEN FLUORESCENT PROTEIN; NI INSECT CELLS; HUMAN INTERLEUKIN-2; PURIFICATION; GLYCANS; FUSION; LINES
URI
http://oasis.postech.ac.kr/handle/2014.oak/25692
DOI
10.1021/BP034375A
ISSN
8756-7938
Article Type
Article
Citation
BIOTECHNOLOGY PROGRESS, vol. 20, no. 4, page. 1192 - 1197, 2004-08
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 HWANG, DONG SOO
Div of Integrative Biosci & Biotech
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