Expression of functional human transferrin in stably transfected Drosophila S2 cells
SCIE
SCOPUS
- Title
- Expression of functional human transferrin in stably transfected Drosophila S2 cells
- Authors
- Lim, HJ; Kim Yeon Kyu; Hwang, DS; Cha Hyung Joon
- Date Issued
- 2004-07
- 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
- https://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, 2004-07
- Files in This Item:
- There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.