Department of Applied Biology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Department of Applied Biology, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Protein Expr Purif. 2022 Dec;200:106147. doi: 10.1016/j.pep.2022.106147. Epub 2022 Jul 31.
Trichosanthin (TCS), as a type 1 ribosome-inactivating protein, has a very high cytoplasmic activity in vitro and can quickly kill cancer cells. However, it is easily filtered and cleared by the kidney, which results in the short half-life and severely limits its application. In this study, we constructed several recombinant proteins by fusing the albumin binding domain mutant ABD035(abbreviated as ABD) to the N- or C-terminus of TCS to endow the recombinant TCS fusion protein with a longer half-life property binding with endogenous human serum albumin (HSA) via ABD to effectively exert its anti-tumor activity in vivo. Pull down, Dynamic light scattering and ELISA assays all showed that TCS fused with two ABD sequences at the C-terminus of TCS, has stronger binding capacity to HSA in vitro than TCS with one ABD. In vivo studies in BALB/C mice were performed and the elimination half-life of TCS-ABD-ABD is about 15-fold longer compared to TCS and anti-tumor activity is about 30% higher than that of TCS alone in BALB/C mouse experiments. Moreover, we found that TCS with two ABDs in tandem have the highest soluble expression level, more than 5 times higher than that of TCS, and the yield of purified protein of TCS-ABD-ABD was as high as 68.9 mg/L culture solution, which was about 7-fold higher than that of TCS. Furthermore, MTT assay showed that the anti-tumor activity of TCS-ABD-ABD was significantly higher than TCS fused with only one ABD sequence, indicating that the repeated ABD sequences facilitated the biological activity of TCS. In this paper, the fusion of the albumin-binding domain in tandem with TCS can effectively improve its stability in vivo and also significantly increase its soluble expression, expanding the application of the albumin-binding domain in the high soluble expression and stability of protein drugs.
天花粉蛋白（TCS）作为一种 1 型核糖体失活蛋白，在体外具有非常高的细胞质活性，能够快速杀死癌细胞。然而，它很容易被肾脏过滤和清除，导致半衰期短，严重限制了其应用。在本研究中，我们通过将白蛋白结合域突变体 ABD035（简称 ABD）融合到 TCS 的 N 端或 C 端，构建了几种重组蛋白，使重组 TCS 融合蛋白与内源性人血清白蛋白（HSA）通过 ABD 结合具有更长的半衰期特性，从而有效地在体内发挥其抗肿瘤活性。拉下实验、动态光散射和 ELISA 分析均表明，TCS 与两个 ABD 序列在 C 端融合后，与 TCS 相比，在体外与 HSA 的结合能力更强。在 BALB/C 小鼠体内研究中，TCS-ABD-ABD 的消除半衰期约比 TCS 长 15 倍，在 BALB/C 小鼠实验中的抗肿瘤活性比 TCS 单独使用高约 30%。此外，我们发现串联的两个 ABD 具有最高的可溶性表达水平，比 TCS 高 5 倍以上，TCS-ABD-ABD 的纯化蛋白产量高达 68.9 mg/L 培养液，比 TCS 高 7 倍。此外，MTT 分析表明，TCS-ABD-ABD 的抗肿瘤活性明显高于仅融合一个 ABD 序列的 TCS，表明重复 ABD 序列促进了 TCS 的生物学活性。在本文中，白蛋白结合域与 TCS 的串联融合可以有效地提高其体内稳定性，同时也显著提高其可溶性表达，扩大了白蛋白结合域在蛋白质药物的高可溶性表达和稳定性中的应用。
