Effective expression and purification of bioactive recombinant soluble LIGHT.
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.
Biomolecular Research Laboratories Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
Isamu
I
Tsuji
Keiji
K
Iwamoto
Yasushi
Y
Shintani
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D000818:Animals; D017209:Apoptosis; D017122:Bacteriophage T4 / Q000737:chemistry / Q000378:metabolism; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies; D051379:Mice; D017510:Protein Folding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; D014764:Viral Proteins; C057839:fibritin protein, Enterobacteria phage T4; D003545:Cysteine