# High yield purification of an isoleucine zipper-modified CD95 ligand for efficient cell apoptosis initiation and with biotin or DNA-oligomer binding domain to probe ligand functionalization effects

## TL;DR

This paper describes a new method to purify and modify CD95L, a protein that triggers cell death, to make it more effective for biomedical applications.

## Contribution

A novel isoleucine zipper-modified CD95L with enhanced apoptosis efficiency and functionalization capabilities is developed.

## Key findings

- IZ-CD95L shows high apoptosis initiation efficiency when crosslinked with antibodies.
- A fast and efficient production and purification workflow for CD95L is established.
- The stabilized trimeric structure of CD95L is identified as the most potent inducer of apoptosis.

## Abstract

Cluster 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. However, CD95L, when presented in solution, exhibits a low efficiency to induce apoptosis signaling in human cells.

Here, 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 initiation efficiency. This efficiency is further enhanced by antibody-mediated crosslinking of IZ-CD95L.A cysteine amino acid fused behind the IZ is used as a versatile coupling site for bionanotechnological applications or for the development of biomedical assays. A fast, cheap, and efficient 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.

The 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, potentially, anticancer therapy.

The online version contains supplementary material available at 10.1186/s12896-025-00986-2.