Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery.
Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery.
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DOI：
10.1016/j.bbamem.2010.02.003
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发表时间：
2010-12
期刊：
影响因子：
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通讯作者：
Hao‐Hsin Yu;I. Nakase;S. Pujals;Hisaaki Hirose;Gen Tanaka;Sayaka Katayama;M. Imanishi;S. Futaki
中科院分区：
文献类型：
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作者：
Hao‐Hsin Yu;I. Nakase;S. Pujals;Hisaaki Hirose;Gen Tanaka;Sayaka Katayama;M. Imanishi;S. Futaki
表达蛋白连接(EPL)是蛋白质与其他蛋白质或多肽进行天然化学连接的一种有效方法。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(CPPs)融合蛋白用于细胞内递送的可行性。利用内含素介导的亲和甲壳素结合标签(IMPACT)系统进行纯化，制备了EGFP硫酯(EGFP-SR)。优化了EGFP-SR与精氨酸12聚体(R12)的连接条件，获得了高产的融合蛋白。EPL程序还允许通过在与R12肽连接之前满意地去除EGFP-SR的ET来制备含有低水平内毒素(ET)的EGFP-R12。EPL制备的EGFP与R12及其d-异构体的融合蛋白与直接在Escherichiacoli中表达的融合蛋白相比，具有相似的细胞摄取水平。
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.