High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction
Shang, X.; Bartels, N.; Weck, J. M.; Suppmann, S.; Basquin, J.; Heuer-Jungemann, A.; Monzel, C.
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BackgroundCluster of differentiation 95 (CD95/Fas/Apo1) as part of the Tumor-necrosis factor (TNF) receptor family is a prototypic trigger of the extrinsic apoptotic pathway and its activation by the trimeric ligand CD95L is of high interest for anticancer therapy. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells. ResultsHere, we design a recombinant CD95L exhibiting an isoleucine zipper (IZ) motif at the N-terminus for stabilization of the trimerized CD95L and demonstrate its high apoptosis induction efficiency. A cysteine amino acid fused behind the IZ is further used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and high-yield production of CD95L via the HEK293T secretory expression system is presented, along with CD95L affinity purification and functionalization. We verified the biological activity of the purified protein and identified a stabilized trimeric CD95L structure as the most potent inducer of apoptosis signaling. ConclusionsThe workflow and the findings reported here will streamline a wide array of future low- or high-throughput TNF-ligand screens, and their modification towards improving apoptosis induction efficiency and anticancer therapy.
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