Prokaryotic expression and characterization of IL-21 and its combined effect with IL-2 as an efficacious chimeric drug for cancer therapy

Abstract Human interleukin-2 (IL-2) and interleukin-21 (IL-21) are pleiotropic cytokines crucial for immune cell activation and proliferation. This study reports the cloning, expression, and characterization of IL-21 in E. coli system using the pET28a (+) vector. Optimal induction with 0.1 mM IPTG yielded ~ 40% IL-21 expression in inclusion bodies. Purification via metal affinity chromatography followed by urea-gradient refolding recovered ~ 15% protein with ~ 95% purity from 1 L culture. MTT assays demonstrated dose- and time-dependent cytotoxicity of IL-21, IL-2, and their combination against HepG-2, MCF-7, and Jurkat cell lines over 48 h. IL-21 exhibited the strongest cytotoxic effect, producing significantly lower LC₅₀ values ( p < 0.05) than IL-2 in HepG2 and Jurkut cell lines while IL-2 exhibited strongest effect in MCF-7 cell line. Data are presented as mean ± SD from three independent experiments. Based on these findings, an in silico chimeric fusion protein (IL-21 linked to IL-2 via a flexible linker) was designed. Structural predictions indicated, 52.83% helices, 11.64% strands, 4.4% turns, and 31.13% coils, with two domains of 153 amino acids each. Docking analysis revealed strong receptor interactions through hydrogen bonding, while molecular dynamics simulations (100 ns, 300 K) confirmed structural stability with minimal backbone fluctuations. The fused system successfully equilibrated with a stable average RMSD of 0.45 ± 0.03 nm and a tight, compact conformation (R g = 2.14 ± 0.02 nm), sustained by a dense network of over 184 average hydrogen bonds. Collectively, these findings highlight the translational promise of the IL-21/IL-2 fusion construct as a potential cytokine-based anticancer therapeutic. Future studies will focus on experimental expression of the fusion protein and in vitro validation of its structural integrity, receptor interactions, immunostimulatory activity, and antitumor efficacy.