Genetically Encoded Protease Substrate Based on Lanthanide-Binding Peptide for Time-Gated Fluorescence Detection

Abstract

The study of biomolecular interactions is at the heart of biomedical research. Fluorescence and Förster resonance energy transfer (FRET) are potent and versatile tools in studying these interactions. Fluorescent proteins enable genetic encoding which facilitates their use in recombinant protein and in vivo applications. To eliminate the autofluorescence background encountered in applications based on fluorescent proteins, lanthanide labels can be used as donor fluorophores. Their long emission lifetime enables the use of time-gating that significantly improves assay sensitivity. In this work, we have combined the favorable characteristics of a terbium-ion-containing lanthanide-binding peptide (Tb(3+)-LBP) and green fluorescent protein (GFP) in a FRET-based homogeneous protease activity assay. The used genetically engineered construct had LBP and GFP sequences at adjacent ends of a linker that encoded the recognition sequence for caspase-3. Caspase proteases are central mediators in apoptosis and, consequently, are of great interest in the pharmaceutical industry. The designed fluorogenic protease substrate was applied for the detection of caspase-3 activity. We were able to demonstrate, for the first time, the applicability of a Tb(3+)-LBP-GFP energy-transfer pair in a protease activity assay. The intrinsically fluorescent and genetically encodable components enable easy expression of the construct without the need of cumbersome chemical labeling. By varying the fluorescent protein and the protease specificity of the internal linker sequence, the method can be applied for the detection of a wide variety of proteases.

Lanthanide light for biology and medical diagnosis

“…Excitation of Tb III through tryptophan moieties results in FRET to GFP that consequently emits at 520 nm with a lifetime equal to that of Tb III . Upon interaction with the enzyme, the linker is destroyed, FRET is inhibited and sensitized emission decreases dramatically [45].…”

Application of lanthanide luminescence in probing enzyme activity

“…82 More Please do not adjust margins Please do not adjust margins recently, Hamachi and coworkers have used a known Tb(III) binding site along with fluorescein-labelling in the mannose binding protein, Concanavalin A, to develop a FRET assay for tracking the enzymatic hydrolysis of mannose from a glycoprotein by α-mannosidase. 85 The Imperiali group have worked extensively on the development of lanthanide binding tags (LBTs), 15,45,[86][87][88][89][90][91][92] comprising between 17 and 23 amino acids which can coordinate to lanthanide(III) ions, predominantly via aspartate and glutamate residues. Typically, the peptide contains a tryptophan residue to sensitise a coordinated Tb(III) ion.…”

“…Lanthanide binding tags have been utilised to monitor protease activity. 92 A genetically encoded protein consisting of a green fluorescent protein (GFP) and an LBT, separated by a linker containing cleavage sites for the proteases caspase and trypsin ( Figure 5a). When the protein construct is intact, longlived FRET occurs between the Tb(III) donor (present in the LBT) and the proximate GFP acceptor.…”

“…a) Detection of protease activity by FRET from the LBT to GFP. Upon proteolytic cleavage at the protease recognition sequence, the LBT and GFP are no longer part of the same construct so FRET does not occur, and Tb(III) emission is observed92 . b) Protein kinase inducible domain,99 phosphorylation of an amino acid (X) generates a Tb(III) ion chelating peptide, creating a luminescent complex.…”

A Label-free Time-resolved Luminescent Platform for Sensitive Endonuclease V Detection Based on Exonuclease III Regulated DNA-Tb3+ Luminescence

“…[16][17][18][19] Trivalent lanthanide ions (Ln 3+ ), such as terbium ion (Tb 3+ ), are always chosen to generate TRL in bioassays for their superior performance (sharply spiked emission bands, large Stokes-shifts, and long lifetimes). [20][21][22] Due to their abundant 4f semi-stable state energy level, as well as the particular transition characteristics, they could possess a long-lived excited state, which will bring out delayed luminescence. Owing to the f-f transition is spin-forbidden, it is difficult to direct excitation of Ln 3+ to luminesce.…”