Matsumoto Rena, Niwa Tatsuya, Kuno Kaori, Shimane Yasuhiro, Kuruma Yutetsu, Kanamori Takashi
GeneFrontier Corporation, 273-1 Kashiwa, Kashiwa, Chiba 277-0005, Japan.
Cell Biology Center, Institute of Integrated Research, Institute of Science Tokyo, Yokohama, Kanagawa 226-8501, Japan.
ACS Synth Biol. 2025 Jul 18;14(7):2729-2738. doi: 10.1021/acssynbio.5c00155. Epub 2025 Jun 19.
Post-translational modifications are an essential process for proper protein function and localization. In particular, lipid modification plays a crucial role in the spatial regulation of proteins functioning on a lipid membrane surface. While cell-free protein synthesis allows rapid protein production, technical advances in lipidation modification are behind. Here, we developed a cell-free system for the myristoylation and palmitoylation of proteins. Based on our previous study, we improved myristoylation efficiency by trimming a precursor nascent peptide, which undergoes lipidation at the N-terminal glycine. We also found that N-myristoyltransferase (NMT) catalyzes both myristoylation and palmitoylation. The localization of lipidated proteins onto liposomes is further aided by the insertion of polyarginine residues downstream of the NMT recognition site. Finally, we demonstrated that lipidation of VHH antibodies and localization onto liposomes resulted in target-specific binding to cancer cells. This system offers a platform for displaying soluble proteins on lipid membranes, with potential applications in developing liposomes for targeted cell binding.
翻译后修饰是蛋白质正常功能和定位的重要过程。特别是，脂质修饰在脂质膜表面发挥作用的蛋白质的空间调节中起着关键作用。虽然无细胞蛋白质合成能够快速生产蛋白质，但脂质化修饰的技术进展却较为滞后。在此，我们开发了一种用于蛋白质肉豆蔻酰化和棕榈酰化的无细胞系统。基于我们之前的研究，我们通过修剪在前体新生肽的N端甘氨酸处发生脂质化的前体新生肽来提高肉豆蔻酰化效率。我们还发现N-肉豆蔻酰转移酶（NMT）催化肉豆蔻酰化和棕榈酰化。NMT识别位点下游插入多聚精氨酸残基进一步有助于脂质化蛋白质定位到脂质体上。最后，我们证明VHH抗体的脂质化以及在脂质体上的定位导致其与癌细胞的靶向特异性结合。该系统提供了一个在脂质膜上展示可溶性蛋白质的平台，在开发用于靶向细胞结合的脂质体方面具有潜在应用。
