Format

Send to

Choose Destination
PLoS One. 2016 Jan 21;11(1):e0147553. doi: 10.1371/journal.pone.0147553. eCollection 2016.

Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It.

Author information

1
Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.

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.

PMID:
26793973
PMCID:
PMC4721919
DOI:
10.1371/journal.pone.0147553
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center