Ghosh Souvik, Birke Ramona, Natarajan Ashwin Karthick, Broichhagen Johannes
Leibniz-Forschungsinstitut Für Molekulare Pharmakologie (FMP), Berlin, Germany.
Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany.
J Pept Sci. 2025 May;31(5):e70015. doi: 10.1002/psc.70015.
Self-labelling proteins like SNAP- and HaloTag have advanced imaging in life sciences by enabling live-cell labeling with fluorophore-conjugated substrates. However, the typical one-fluorophore-per-protein system limits signal intensity. To address this, we developed a strategy using the ALFA-tag system, a 13-amino acid peptide recognized by a bio-orthogonal and fluorescently labelled nanobody, for signal amplification. We synthesized a pentavalent ALFA peptide and used an azidolysine for conjugation with a Cy5-modified SNAP- or HaloTag ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, and the peptides covalently reacted with their respective tag. HEK293 cells expressing SNAP- and HaloTag-mGluR2 fusion proteins were labeled with ALFA-Cy5 substrates, and confocal microscopy revealed a significant enhancement in the far-red signal intensity upon nanobody addition, as quantified by integrated signal density ratios. Comparisons between SNAP- and HaloTag substrates showed superior performance for the latter, achieving better signal-to-noise and signal-to-background ratios, as well as overall signal intensity in plasma membrane-localized regions. Our results demonstrate the potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This strategy combines the photostability of synthetic fluorophores with multivalent labeling, providing a powerful tool for advanced imaging applications including super-resolution in cells. Its versatility is expandable across diverse protein systems and colors.
像SNAP标签和HaloTag这样的自标记蛋白通过使用荧光团偶联底物进行活细胞标记，推动了生命科学领域的成像技术发展。然而，典型的每个蛋白一个荧光团的系统限制了信号强度。为了解决这个问题，我们开发了一种策略，使用ALFA标签系统（一种由生物正交且荧光标记的纳米抗体识别的13个氨基酸的肽）来放大信号。我们合成了一种五价ALFA肽，并使用叠氮赖氨酸通过应变促进的点击化学与Cy5修饰的SNAP或HaloTag配体进行偶联。SDS-PAGE的体外测量显示了标记情况，并且这些肽与其各自的标签发生了共价反应。用ALFA-Cy5底物标记表达SNAP和HaloTag-mGluR2融合蛋白的HEK293细胞，共聚焦显微镜显示添加纳米抗体后远红光信号强度显著增强，通过积分信号密度比进行量化。SNAP和HaloTag底物之间的比较表明，后者具有更好的性能，在质膜定位区域实现了更好的信噪比和信号背景比以及整体信号强度。我们的结果证明了基于ALFA标签的系统放大自标记蛋白荧光信号的潜力。这种策略将合成荧光团的光稳定性与多价标记相结合，为包括细胞超分辨率在内的先进成像应用提供了一个强大的工具。其通用性可扩展到各种蛋白质系统和颜色。
