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. 2024 Aug 6;720:150097. doi: 10.1016/j.bbrc.2024.150097. Epub 2024 May 9.
Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.
内含子是独特的单 turnover 酶，无需任何外部辅助因子或能量即可从前体蛋白中自我切除。在大多数情况下，内含子与外显子序列共价连接，蛋白质剪接自发发生。在这项研究中，开发了一种基于两个非典型分裂内含子的新型蛋白质连接系统，它们之间没有交叉反应，其中一个 S1 和一个 S11 分裂内含子融合蛋白的大片段充当蛋白质连接酶，小片段（只有几个氨基酸长）分别融合到 N-外显子和 C-外显子上。拼接活性在大肠杆菌和体外不同的外显子序列中得到了证明，体外的拼接效率约为 15%。进一步优化了体外蛋白质转剪接，并探讨了可能的反应解释。作为概念验证，我们期望这种方法扩展基于转剪接的蛋白质工程的范围，并为基于内含子的蛋白质连接酶提供新的线索。
