College of Biological Science and Medical Engineering, Donghua University, Shanghai, 201620, PR China.
College of Biological Science and Medical Engineering, Donghua University, Shanghai, 201620, PR China.
Biochem Biophys Res Commun. 2023 May 7;655:44-49. doi: 10.1016/j.bbrc.2023.03.023. Epub 2023 Mar 10.
Conditional protein splicing is a powerful biotechnological tool that can be used to post-translationally control the activity of target proteins. Here we demonstrated a novel conditional protein splicing approach in which the small ubiquitin-like modifier (SUMO) protease induced the splicing of an atypical split intein. The engineered Ter DnaE-3 S11 split intein which has a small C-intein segment with only 6 amino acids was used in this study. A SUMO tag was fused to the N-terminus of the C-intein to inhibit the protein trans-splicing in vitro. The splicing products could be detected in 15 min with the addition of SUMO protease by western blotting and the splicing efficiency was ∼4-fold higher than the control without SUMO protease for overnight reaction. This engineered Ter DnaE-3 S11 split intein-mediated protein trans-splicing had been further shown to be triggered by SUMO protease in different exteins in vitro. Our study provides new insights into the regulation of protein splicing and is a promising tool for the control of protein structure and function in vitro.
条件性蛋白剪接是一种强大的生物技术工具，可用于对靶蛋白的活性进行翻译后调控。在此，我们展示了一种新型的条件性蛋白剪接方法，其中小泛素样修饰物 (SUMO) 蛋白酶诱导非典型分裂整合酶的剪接。本研究中使用了经过工程改造的 Ter DnaE-3 S11 分裂整合酶，它具有仅 6 个氨基酸的小 C-整合酶片段。SUMO 标签融合到 C-整合酶的 N 端，以抑制体外蛋白质的转剪接。通过 Western blot 检测到加入 SUMO 蛋白酶 15 分钟后即可检测到剪接产物，并且与没有 SUMO 蛋白酶的过夜反应相比，剪接效率提高了约 4 倍。该工程化的 Ter DnaE-3 S11 分裂整合酶介导的蛋白质转剪接已进一步显示可在体外不同外显子中被 SUMO 蛋白酶触发。我们的研究为蛋白质剪接的调控提供了新的见解，是体外控制蛋白质结构和功能的有前途的工具。
