Document Detail


Expression of functional human transferrin in stably transfected Drosophila S2 cells.
MedLine Citation:
PMID:  15296447     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) 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 microg/mL, producing 6.8 microg/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 His6-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.
Authors:
Hye Jung Lim; Yeon Kyu Kim; Dong Soo Hwang; Hyung Joon Cha
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biotechnology progress     Volume:  20     ISSN:  8756-7938     ISO Abbreviation:  Biotechnol. Prog.     Publication Date:    2004 Jul-Aug
Date Detail:
Created Date:  2004-08-06     Completed Date:  2005-02-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8506292     Medline TA:  Biotechnol Prog     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1192-7     Citation Subset:  IM    
Affiliation:
Department of Chemical Engineering and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
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MeSH Terms
Descriptor/Qualifier:
Animals
Blotting, Western
Cell Line
Chromatography, Affinity
Drosophila
Electrophoresis, Gel, Two-Dimensional
Electrophoresis, Polyacrylamide Gel
Genetic Vectors
Glycosylation
Humans
Recombinant Proteins / genetics,  isolation & purification,  metabolism
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Transfection
Transferrin / genetics*,  isolation & purification,  metabolism,  physiology
Chemical
Reg. No./Substance:
0/Recombinant Proteins; 11096-37-0/Transferrin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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