Lee Tae Hee, Kim Kwang Soo, Kim Jin Hee, Jeong Jae-Ho, Woo Hye Ryun, Park So Ra, Sohn Myung-Ho, Lee Hyeon Ju, Rhee Joon Haeng, Cha Sun-Shin, Hwang Joo-Hee, Chung Kyung Min
Department of Microbiology and Immunology, Jeonbuk National University Medical School, Jeonju, Jeonbuk 54896, Republic of Korea; Institute for Medical Science, Jeonbuk National University Medical School, Jeonju, Jeonbuk 54896, Republic of Korea.
Department of Microbiology, Chonnam National University Medical School, Hwasun-gun 58128, Republic of Korea; Combinatorial Tumor Immunotherapy Medical Research Center, Chonnam National University Medical School, Hwasun-gun 58128, Republic of Korea.
J Immunol Methods. 2020 Apr;479:112750. doi: 10.1016/j.jim.2020.112750. Epub 2020 Jan 22.
The specific recognition between a monoclonal antibody (mAb) and its epitope can be used in a tag system that has proved valuable in a wide range of biological applications. Herein, we describe a novel tag called RA-tag that is composed of a seven amino acid sequence (DIDLSRI) and recognized by a highly specific mAb, 47RA, against the bacterial toxin Vibrio vulnificus RtxA1/MARTX. By using recombinant proteins with the RA-tag at the N-terminal, C-terminal, or an internal site, we demonstrated that the tag system could be an excellent biological system for both protein purification and protein detection in enzyme-linked immunosorbent, Western blot, flow cytometry, and immunofluorescence staining analyses in Escherichia coli, mammalian cell lines, yeast, and plant. In addition, our RA-tag/47RA mAb combination showed high sensitivity and reliable affinity (K = 5.90 × 10 M) when compared with conventional tags. Overall, our results suggest that the RA-tag system could facilitate the development of a broadly applicable tag system for biological research.
单克隆抗体（mAb）与其表位之间的特异性识别可用于一种标签系统，该系统已被证明在广泛的生物学应用中具有重要价值。在此，我们描述了一种名为RA标签的新型标签，它由一个七氨基酸序列（DIDLSRI）组成，并被一种高度特异性的单克隆抗体47RA识别，该抗体针对细菌毒素创伤弧菌RtxA1/MARTX。通过使用在N端、C端或内部位点带有RA标签的重组蛋白，我们证明该标签系统在大肠杆菌、哺乳动物细胞系、酵母和植物的酶联免疫吸附测定、蛋白质印迹、流式细胞术和免疫荧光染色分析中，对于蛋白质纯化和蛋白质检测而言可能是一个出色的生物学系统。此外，与传统标签相比，我们的RA标签/47RA单克隆抗体组合表现出高灵敏度和可靠的亲和力（K = 5.90×10⁻⁸ M）。总体而言，我们的结果表明，RA标签系统可能有助于开发一种广泛适用于生物学研究的标签系统。
