State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
Appl Microbiol Biotechnol. 2022 Feb;106(4):1511-1520. doi: 10.1007/s00253-022-11807-3. Epub 2022 Feb 8.
Recombinant protein pharmaceutical agents have been widely used for cancer treatment. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has broad-spectrum antitumor activity, its clinical applications are limited because most tumor cells eventually develop resistance to TRAIL-induced apoptosis through various pathways. Prostate apoptosis response-4 (Par-4) selectively induces apoptosis in cancer cells after binding to the cell surface receptor, GRP78. In this study, TRAIL was fused with the core domain of Par-4 (SAC) to produce a novel recombinant fusion protein. To obtain solubly expressed fusion protein, a small ubiquitin-related modifier (SUMO) was added to the N-terminus of the target protein. Cytotoxicity assays showed that the purified fusion protein exhibited more significant antitumor activity on cancer cells than that by native TRAIL. The connection order and linker sequence of the fusion proteins were optimized. In vitro cytotoxicity assay showed that the SAC-TRAIL fusion protein, which contained a flexible linker (GS), optimally inhibited the proliferation of cancer cells. Immunofluorescence assays demonstrated that SAC-TRAIL could efficiently and specifically bind to cancer cells. Additionally, circular dichroism assays showed that the secondary structure of the recombinant protein with a flexible linker (GS) has both a lower α-helix and higher random coiling, which facilitates the specific binding of SAC-TRAIL to the receptor. Collectively, these results suggest that the novel recombinant fusion protein SAC-(GS)-TRAIL is a potential therapeutic agent for cancer. KEY POINTS: • Improved tumor growth suppression and apoptosis induction potency of SAC-TRAIL. • Enhanced targeting selectivity of SAC-TRAIL in cancer cells. • Lower α-helix and higher random coiling in SAC-TRAIL with flexible linker (GS).
重组蛋白药物已被广泛用于癌症治疗。虽然肿瘤坏死因子相关凋亡诱导配体（TRAIL）具有广谱抗肿瘤活性，但由于大多数肿瘤细胞最终通过各种途径对 TRAIL 诱导的细胞凋亡产生耐药性，其临床应用受到限制。前列腺凋亡反应蛋白 4（Par-4）与细胞表面受体 GRP78 结合后，选择性诱导癌细胞凋亡。在本研究中，TRAIL 与 Par-4 的核心结构域（SAC）融合，产生了一种新型重组融合蛋白。为了获得可溶表达的融合蛋白，在靶蛋白的 N 端添加了一个小泛素相关修饰物（SUMO）。细胞毒性试验表明，与天然 TRAIL 相比，纯化的融合蛋白对癌细胞具有更强的抗肿瘤活性。优化了融合蛋白的连接顺序和连接肽序列。体外细胞毒性试验表明，含有柔性连接肽（GS）的 SAC-TRAIL 融合蛋白能最佳抑制癌细胞的增殖。免疫荧光试验表明 SAC-TRAIL 能有效地特异性结合癌细胞。此外，圆二色性试验表明，具有柔性连接肽（GS）的重组蛋白的二级结构具有较低的α-螺旋和较高的无规卷曲，这有利于 SAC-TRAIL 与受体的特异性结合。综上所述，这些结果表明新型重组融合蛋白 SAC-（GS）-TRAIL 是一种有潜力的癌症治疗药物。关键点：•提高了 SAC-TRAIL 的肿瘤生长抑制和凋亡诱导效力。•增强了 SAC-TRAIL 在癌细胞中的靶向选择性。•具有柔性连接肽（GS）的 SAC-TRAIL 的α-螺旋更低，无规卷曲更高。
