Minamihata Kosuke, Maeda Yasukazu, Yamaguchi Satoshi, Ishihara Wataru, Ishiwatari Akira, Takamori Satoshi, Yamahira Shinya, Nagamune Teruyuki
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Biosci Bioeng. 2015 Dec;120(6):630-6. doi: 10.1016/j.jbiosc.2015.04.001. Epub 2015 Apr 29.
Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells.
将外源蛋白质导入细胞能够精确研究导入的蛋白质对细胞功能的影响。因此，在各个研究领域都非常需要能够确定且高通量地控制蛋白质释放到细胞质中的蛋白质转导技术。在本研究中，提出了一种用光敏剂和细胞穿透肽（CPP）标记的链霉亲和素（SA）作为光控蛋白质转导的纳米载体。SA用生物素化的寡聚精氨酸肽（Rpep）进行修饰，Rpep用Alexa Fluor 546（AF546）进行功能化，以实现细胞穿透和内体逃逸功能。SA-Rpep复合物能有效地内化到活的HeLa细胞中，这与Rpep的长度和修饰数量相对应。通过照射AF546的激发光，实现了与由15个精氨酸残基组成的三个以上等摩尔AF546修饰的Rpep共轭的SA在整个细胞质中扩散且无细胞毒性。经证实，优化后的纳米载体能够在光照射区域将生物素化的模型货物蛋白——与硫氧还蛋白融合的增强型绿色荧光蛋白（tEGFP）转导到细胞质中。结果提供了原理证明，即具有多个AF546修饰的Rpep的SA有潜力成为一种通用且便捷的载体，用于将蛋白质光控转导到哺乳动物细胞的细胞质中。
