Abstract
LIGHT (TNFSF14, CD258) is a member of the tumor necrosis factor (TNF) ligand superfamily, which is involved in innate and adaptive immune responses as well as in regulation of cell survival and proliferation. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. In this study, we established an effective strategy for producing bioactive soluble forms of human LIGHT (s-hLIGHT), which is an extracellular region (Ile84-Val240) of human LIGHT (hLIGHT), and soluble forms of mouse LIGHT (s-mLIGHT), which is an extracellular region (Asp72-Val239) of mouse LIGHT (mLIGHT). To enhance the refolding of s-hLIGHT from inclusion bodies in Escherichia coli, we added l-cysteine to the denaturation buffer, which significantly improved the refolding efficiency of s-hLIGHT. However, there was little information available about the biological activity of mLIGHT in the literature because of the difficulty in producing bioactive s-mLIGHT. We produced trimeric s-mLIGHT by fusing s-mLIGHT with a trimerization domain termed “foldon” from bacteriophage T4 fibritin (Foldon-mLIGHT). We further demonstrated that Foldon-mLIGHT inhibited the growth of mouse carcinoma cells at the picomolar range, indicating that trimerization of s-mLIGHT is essential for its biological activity.
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Acknowledgments
The authors thank Drs. Ito T, Matsui H, Kurokawa T, and Taniyama Y for their kind guidance, constructive discussions, and encouragement throughout this work.
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Tsuji, I., Iwamoto, K., Shintani, Y. (2014). Effective Expression and Purification of Bioactive Recombinant Soluble LIGHT. In: Bayry, J. (eds) The TNF Superfamily. Methods in Molecular Biology, vol 1155. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0669-7_17
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DOI: https://doi.org/10.1007/978-1-4939-0669-7_17
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