Yu Hao-Hsin, Nakase Ikuhiko, Pujals Sílvia, Hirose Hisaaki, Tanaka Gen, Katayama Sayaka, Imanishi Miki, Futaki Shiroh
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochim Biophys Acta. 2010 Dec;1798(12):2249-57. doi: 10.1016/j.bbamem.2010.02.003. Epub 2010 Feb 17.
Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.
表达蛋白连接（EPL）是一种将蛋白质与其他蛋白质或肽进行天然化学连接的有用方法。本研究评估了EPL在制备增强型绿色荧光蛋白（EGFP）与化学合成的细胞穿透肽（CPP）的融合蛋白用于细胞内递送方面的实用性。使用带有亲和几丁质结合标签（IMPACT）系统的内含肽介导纯化方法，制备了EGFP的硫酯（EGFP-SR）。优化EGFP-SR与精氨酸12聚体（R12）的连接反应，可高产率地获得融合蛋白。EPL程序还可以通过在与R12肽连接之前对EGFP-SR进行令人满意的内毒素（ET）去除，来制备含有低水平内毒素（ET）的EGFP-R12。与直接在大肠杆菌中表达的融合蛋白相比，通过EPL制备的EGFP与R12以及R12的d-异构体的融合蛋白显示出相似水平的细胞摄取。
