Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.
Bibliographical noteFunding Information:
We are grateful to Dr. Sungchul Hohng (Seoul National University) for single molecule data analysis. We also thank Dr. Sang-Hyeon Kang (iNtRON Biotechnology) and Dr. Dong-Hwa Shon (Korea Food Research Institute) for technical supports and advice. The expression vector encoding the human initiator tRNA gene was a gift from Dr. Sung Hun Kim (Seoul National University). We also thank Dr. Joo Hee Chung of the KBSI (Seoul) for HPLC and MALDI TOF analysis. This work was supported in part by the Brain Korea 21 (BK21) PLUS program (J.K.) and the Grant HI13C0826 from MoHW (Ministry of Health and Welfare) and MAFRA 716002-7 from MFARA (Ministry of Agriculture, Food and Rural Affairs) of Korean Government (B.L.S.).
All Science Journal Classification (ASJC) codes