title: Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag.
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.
authors.affiliation: Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Watanabe
authors.forename: Shin
authors.initials: S
authors.lastname: Mizukami
authors.forename: Yuri
authors.initials: Y
authors.lastname: Akimoto
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Hori
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D009154:Mutation; D010406:Penicillins / Q000737:chemistry*; D011355:Prodrugs / Q000737:chemistry*; D011994:Recombinant Proteins; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010406:Penicillins; D011355:Prodrugs; D011994:Recombinant Proteins; D001618:beta-Lactamases