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  • Title: Effective expression and purification of bioactive recombinant soluble LIGHT.
    Author: Tsuji I, Iwamoto K, Shintani Y.
    Journal: Methods Mol Biol; 2014; 1155():201-13. PubMed ID: 24788184.
    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 (Ile(84)-Val(240)) of human LIGHT (hLIGHT), and soluble forms of mouse LIGHT (s-mLIGHT), which is an extracellular region (Asp(72)-Val(239)) 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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