Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Shuji Watanabe, Shin Mizukami, Yuri Akimoto, Yuichiro Hori, Kazuya Kikuchi

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

Abstract

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.

Original languageEnglish
Pages (from-to)8342-8349
Number of pages8
JournalChemistry - A European Journal
Volume17
Issue number30
DOIs
Publication statusPublished - 2011 Jul 18
Externally publishedYes

Keywords

  • fluorescent probes
  • microscopy
  • prodrugs
  • protein labeling
  • β-lactamase

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry

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