Life science
Medical & healthcare, Drug development and diagnosis

Development of versatile fluorescent protein labeling probes
for live-cell imaging
Department of Applied Chemistry, Graduate School of Engineering
Professor Kazuya Kikuchi			https://researchmap.jp/kikuchilab315?lang=en

Associate Professor Masafumi Minoshima

https://researchmap.jp/cbatgcm_m33?lang=en

Life science

Abstract
We herein developed a versatile chemical protein-labeling tool
based on fluorophore-conjugated diazabicyclooctane β-lactamase inhibitors (BLIs) and wild-type β-lactamase protein tag.
The fluorescent probes efficiently formed a stable complex withb-lactamase, and the labeled proteins were visualized over a long
period of time in live cells. Moreover, a fluorophore-conjugated BLI
prodrug enabled labeling intracellular proteins due to improved
cell membrane permeability. Lastly, combining the labeling toolwith a pH-activatable fluorescent probe allowed visual monitoringof lysosomal protein translocation during autophagy.

▍ Background & Results

pH-activatable fluorophore (pH-RD4). pH-RD4 increased fluorescence signals in acidic pH corresponding to lysosomal environment. After labeling intracellular protein in live cells with pH-RD4,the cells were starved by replacing the medium with an aminoacid-depleted medium. The time-lapse images revealed that thefluorescence spots were found after two hours in the starvation
medium, and the size and number then increased over time. Thisfinding demonstrated that pH-RD4 is capable of long-term trackingof proteins and imaging the pH environment during the autophagy
process.

▍ Significance of the research and Future perspective

In this study, we developed a versatile fluorescent protein labeling!"#$ system using a series of RD probes bearing BLI and its prodReal-time monitoring of protein dynamics and sensing of theirrug. This β-lactamase-based system has excellent protein labelingsurrounding environment are important methods to understand theabilities in living cells, similar to existing protein labeling systems.
function of protein in live cells. Fluorescent labeling tools are thus
Our protein labeling method can serve as a versatile tool for studneeded that possess fast labeling kinetics, high efficiency, andying protein dynamics within living cells, which contributes toward
long-term stability. Our group has developed a covalent protein
advancing our understanding of molecular biology and medicine.
labeling system that uses the catalytically inactive mutant of βlactamase as a protein tag. Meanwhile, the labile β-lactam ring!"#$%&'()*+%)&',(-%$.#/(01&', !2)*3$*-*1%($*,(of the substrates has pronounced chemical susceptibility andpotential dissociation from the protein complex, which limits theprolonged imaging of intracellular protein dynamics. We initially!"#$%$,.$%& !/"0(*010'&conjugated relebactam, a piperidine analogue of BLI, with a te2).2-2*$%
tramethylrhodamine (TAMRA) fluorophore to afford the new probe!"#$%&'($)*
RD for labeling β-lactamase. RD efficiently labeled β-lactamase(!+,)-$).)/%0and formed a stable complex. In addition, RD labeled β-lactamase tag expressing in plasma membrane in living cells. We further!"#$%&'(&)*+,%$-&
developed a membrane-permeable fluorescent probe (RD3) using
3%$*&2)a BLI prodrug. RD3 showed membrane permeability and enabled
intracellular protein labeling over a prolonged period of time in livecells. Then we appliedthe表labeling method to imaging transloca補足図
tion of proteins into lysosomes in response to starvation-induced455)&3*$&#'($#()&6%23%))(
autophagy. Our imaging strategy relied on labeling intracellular&-*,&',($##)
proteins bearing a β-lactamase tag using a labeling probe with aRD（membrane-impermeable）
Membrane protein

pH-RD4（pH-responsive）

RD3（membrane-permeable）
Nuclear protein

Autophagy detection under starvation condition(Lysosomal translocation of protein)

P a t e n t
Minoshima, Masafumi; Umeno, Taro; Kadooka, Kohei et al. Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent
β-Lactamase Inhibitors. Angewandte Chemie International Edition. 2023, 62, e202301704. doi: 10.1002/anie202301704
URL https://www-molpro-mls.eng.osaka-u.ac.jp/English/indexeng/indexENG.html
K e y w o r d fluorescent probe, protein labeling, β-lactamase inhibitor, autophagy
Tr e a t i s e