A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

Abstract

Background: In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used in live-cell applications, partly because it needs to be adjusted to the specific protein under study. Results: We present a generic, 14-residue long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, may be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues. Conclusions: The GCE-tag presented here offers a powerful, easy-to-implement tool for live-cell labeling of cellular proteins with small and bright probes.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

“…To avoid affecting the levels of expression of endogenous NFL, instead of a site-specific TAG knock-in, we introduced the TAG amber codon at the end of the Nefl gene, in the form of a short linker sequence followed by 3x-FLAG. A similar approach was previously used to develop standardized tags for the click labeling of overexpressed proteins 61 . As illustrated in Fig.…”

Softness Meets with Brightness: Dye‐Doped Multifunctional Fluorescent Polymer Particles via Microfluidics for Labeling

“…[71] Usually, a wide range of various fluorescent dyes is utilizing as fluorescent markers owing to their brightness and full color/emission range. [15,22] While molecular labeling is highly useful for diagnosing and tracking the activities of organelles and biomolecules at a much smaller length scale, [72] the labeling with particulate fluorophores allows the imaging of cells, tissues, and extracellular matrix within biological systems through the interfacial interaction-based approach. [73][74] In this regard, particulate fluorophores such as quantum dots, lanthanide-doped nanoparticles, conjugated polymer nanoparticles, fluorescent silica nanoparticles, etc.…”

Using Genetic Code Expansion for Protein Biochemical Studies

“…The authors demonstrated selective and efficient labeling of the target proteins that produced superior readings compared to immunolabeling (Beliu et al, 2019). A similar study utilized bicyclo-non-yne lysine (BCN-Lys) (13) to attach a tetrazine dye to the protein of interest via a N-terminal tag in HEK293T and Cos7 cells (Segal et al, 2020). Interestingly, insertion of BCN-Lys in the N-terminal tag was labeled better than BCN insertion within the target protein.…”

“…In addition, the modified tag was found to be compatible in labeling organelle proteins within their acidic environments. Although the authors demonstrated the ability to switch out the HA-tag with other commonly used tags (Myc and FLAG), the results were not as successful, indicating a need for further optimization (Segal et al, 2020). The ease of simply inserting a tag encoding an ncAA led the authors to state the possibility of genomically inserting the modified tag to monitor endogenously expressed proteins.…”