Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues

Braga Emidio, Nayara; Cheloha, Ross W.

doi:10.1016/j.cbpa.2024.102443

Current Opinion in Chemical Biology

2024

DOI: 10.1016/j.cbpa.2024.102443

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Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues

Nayara Braga Emidio,

Ross W. Cheloha

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2024

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Cited by 4 publications

References 38 publications

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Sortases: structure, mechanism, and implications for protein engineering

Amacher,

Antos

2024

Trends in Biochemical Sciences

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Sortases: structure, mechanism, and implications for protein engineering

Amacher,

Antos

2024

Trends in Biochemical Sciences

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Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies

Basuli,

Shi,

Lindberg

et al. 2024

Bioconjugate Chem.

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Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 ( 18 F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18 F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[ 18 F]-FH 3 RESCA)-NH 2 ) or the unlabeled chelator (H-GGGK((±)-H 3 RESCA)-NH 2 ) followed by labeling with Al[ 18 F]F, respectively. The overall radiochemical yields were 15−43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23−58% (n = 12, decaycorrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400−600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18 F-labeling method to prepare nanobodies (Nb-Al[ 18 F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

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Site-Specific and Fluorescently Enhanced Installation of Post-Translational Protein Modifications via Bifunctional Biarsenical Linker

Antonenko,

Pomorski,

Singh

et al. 2024

ACS Omega

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To understand how particular post-translational modifications (PTMs) affect the function of a target protein, it is essential to first prepare and investigate the target with the modification at the desired position. This drives the continuous development of site-specific protein modification technologies. Here, we present the chemical synthesis and application of the biarsenical linker SrtCrAsH-EDT 2 , which has a dual labeling functionality. This linker, containing a sortase A recognition motif, can be conjugated with any protein containing the LPXTG motif at the C terminus, such as ubiquitin and the SUMO tag, and then attached to a protein of interest (POI) containing a terminal or bipartite (intramolecularly placed) tetracysteine motif. This modification of the POI facilitates the straightforward and rapid incorporation of PTMs, which are further highlighted by the fluorescent biarsenical probe. Consequently, this directly correlates proteins' physical properties and cellular roles under various physiological conditions or in disease states. The proposed one-pot labeling methodology can be utilized to explore the effects of PTMs on proteins, affecting their structure, function, localization, and interactions within the cellular environment. Understanding these effects is crucial for uncovering the complex mechanisms that regulate cellular function and dysfunction.

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Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues

Cited by 4 publications

References 38 publications

Sortases: structure, mechanism, and implications for protein engineering

Sortases: structure, mechanism, and implications for protein engineering

Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies

Site-Specific and Fluorescently Enhanced Installation of Post-Translational Protein Modifications via Bifunctional Biarsenical Linker

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