Faculty of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan.
Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan.
Chembiochem. 2021 Feb 2;22(3):577-584. doi: 10.1002/cbic.202000609. Epub 2020 Nov 6.
Inteins, self-catalytic enzymes, have been widely used in the field of protein engineering and chemical biology. Here, Nostoc punctiforme PCC73102 (Npu) DnaE intein was engineered to have an altered split position. An 11-residue N-intein of DnaE in which Gly and Asp were substituted for Tyr4 and Glu5, respectively, was designed, and the active C-intein variants were acquired by a GFP fluorescence-based screening. The designed N-intein and the obtained active C-intein variants were used to construct a turn-on system for enzyme activities such as human immunodeficiency 1 protease and NanoLuc luciferase. Based on the NanoLuc-intein fusion, we developed two intein pairs, each of which is capable of reacting preferentially, by interchanging the charged amino acids on N- and C-inteins. The specific splicing reactions were easily monitored and discriminated by bioluminescence resonance energy transfer (BRET).
内含子，自我催化的酶，已被广泛应用于蛋白质工程和化学生物学领域。在这里，对来自鱼腥藻 PCC73102 的 DnaE 内含子进行了工程改造，使其具有改变的分裂位置。设计了一个 11 个残基的 DnaE 内含子，其中 Gly 和 Asp 分别取代 Tyr4 和 Glu5，通过 GFP 荧光筛选获得了活性的 C-内含子变体。利用设计的 N-内含子和获得的活性 C-内含子变体构建了人免疫缺陷病毒 1 蛋白酶和 NanoLuc 荧光素酶等酶活性的开启系统。基于 NanoLuc-内含子融合，我们开发了两种内含子对，它们都能够通过交换 N-和 C-内含子上的带电荷氨基酸，优先进行反应。通过生物发光共振能量转移（BRET）可以轻松监测和区分特异性拼接反应。
