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• W2259384772 endingPage "e0147553" @default.
• W2259384772 startingPage "e0147553" @default.
• W2259384772 abstract "TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model." @default.
• W2259384772 created "2016-06-24" @default.
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• W2259384772 date "2016-01-21" @default.
• W2259384772 modified "2023-10-16" @default.
• W2259384772 title "Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It" @default.
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• W2259384772 doi "https://doi.org/10.1371/journal.pone.0147553" @default.
• W2259384772 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4721919" @default.
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• W2259384772 hasPublicationYear "2016" @default.
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