Yayci Abdulkadir, Huang Yen-Hua, Harvey Peta J, Craik David J
Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane QLD, 4072, Australia.
Chembiochem. 2025 Feb 16;26(4):e202400591. doi: 10.1002/cbic.202400591. Epub 2024 Oct 29.
This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self-assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded, and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein-derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein-mediated protein cyclization with potential applications in bioengineering and peptide discovery.
本研究描述了一种用于重组生产环肽的新型条件性内含肽系统的设计、生产和特性。该系统基于两个关键特性：（1）一个通用的外显肽识别位点，可使几乎任何肽环化；（2）内含肽自身内部的一个二级切割位点，能够随意触发剪接。两种内含肽前体经重组表达、纯化，然后在体外自组装以使模型肽卡塔啉B1（kB1）环化。环化的kB1成功纯化、重折叠，并通过质谱和核磁共振进行表征，证明形成了正确的二硫键且结构与合成的kB1相同。重要的是，昆虫细胞毒性试验表明，内含肽衍生的kB1保留了全部生物活性。这项工作建立了一种通用且高效的内含肽介导的蛋白质环化方法，在生物工程和肽发现方面具有潜在应用。
