Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Abstract

Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would profit from more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we selected a number of short tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POIs binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be utilized for POI manipulation in vivo.

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc

“…Recently, small, high affinity protein binders such as nanobodies, single-chain variable fragments (scFvs), Designed Ankyrin Repeat Proteins (DARPins) and others have emerged as versatile tools to fill this gap. By fusing these protein binders to well-characterized protein domains and expressing the fusion proteins in vivo, protein function can be directly manipulated in a predicted manner [1][2][3][4][5] . For example, when a protein functions with multiple parameters, protein binder tools targeting each or a subset of these parameters could help to dissect the requirement of each parameter.…”

Exploring cellular biochemistry with nanobodies

“…The moon tag was used along with an orthogonal epitope recognition pair based on an scFv antibody (named "sun tag") to demonstrate a high degree of stochasticity and variation in translation kinetics in live cells (221). Both the moon tag and the alfa tag system can be used to deliver a nanobody-fluorescent protein fusion to a subcellular site of choice (nucleus, mitochondria, cell membrane) when the nanobodies carry the appropriate targeting signals (223). The orthogonality of the moon tag and alfa tag suggests the potential for multiplexing (223).…”

“…Both the moon tag and the alfa tag system can be used to deliver a nanobody-fluorescent protein fusion to a subcellular site of choice (nucleus, mitochondria, cell membrane) when the nanobodies carry the appropriate targeting signals (223). The orthogonality of the moon tag and alfa tag suggests the potential for multiplexing (223). The VHH05-6E pair can also efficiently redirect subcellular protein localization.…”

Protein visualization and manipulation inDrosophilathrough the use of epitope tags recognized by nanobodies

“…Several nanobodies that recognize small epitope tags have been isolated, including tags known as BC2-tag, EPEA-tag, MoonTag, and ALFA-tag (Traenkle et al, 2015; De Genst et al, 2010 ; Boersma et al, 2019; Tanenbaum et al, 2014; Götzke et al, 2019 ; Cheloha et al, 2020). Some of these have been used to visualize and manipulate tagged proteins by changing their abundance or localization using tag- targeting nanobodies in mammalian cells (Zhao et al, 2019; Vigano et al, 2021). The recently developed ALFA nanobody (NbALFA) recognizes a short peptide of 13 amino acids and provides a system useful for immunoblotting, protein purification, and imaging (fixed or live cells) ( Götzke et al, 2019 ).…”

“…The recently developed ALFA nanobody (NbALFA) recognizes a short peptide of 13 amino acids and provides a system useful for immunoblotting, protein purification, and imaging (fixed or live cells) ( Götzke et al, 2019 ). In addition, it has been recently reported that HA frankenbody, a single-chain variable fragment engineered from anti- HA antibody, can be used for live imaging and protein degradation in vivo in Drosophila (Vigano et al, 2021). However, to the best of our knowledge, short linear epitope tag-specific nanobodies have yet to be applied for use in vivo in Drosophila or other multicellular organisms.…”