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Expression of Functional Human Transferrin in Stably Transfected Drosophila S2 Cells
Author(s) -
Lim Hye Jung,
Kim Yeon Kyu,
Hwang Dong Soo,
Cha Hyung Joon
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp034375a
Subject(s) - recombinant dna , microbiology and biotechnology , chemistry , transfection , biochemistry , affinity chromatography , plasmid , glycan , glycoprotein , gel electrophoresis , molecular mass , biology , gene , enzyme
Human transferrin (hTf) is a serum glycoprotein involved in Fe 3+ 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 μg/mL, producing 6.8 μg/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.