DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Abstract

statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms. 3 AbstractOver the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.protein function in a more controllable fashion (Buckley et al., 2016; Guglielmi et al., 2016; Rost et al., 2017).Recently, another, somewhat different approach has emerged, which allows targeting and manipulating proteins in several ways and in a more systematic manner. Using Jonas Kapp. We also acknowledge the beamline staff at Swiss Light Source (Villigen, Switzerland) for their excellent support.

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

“…We used transient transfection in HeLa cells as a model system to test the binding properties of these protein binders (Brauchle et al, 2014; Moutel et al, 2016; Vigano et al, 2018). We therefore generated mammalian expression constructs for the anti- GCN4 (SunTag) scFv (Tanenbaum et al, 2014), the anti-gp41(MoonTag) nanobody 2H10 (Boersma et al, 2019), the anti-HA scFvs frankenbodies (Zhao et al, 2019) and the anti-ALFA nanobody (Gotzke et al, 2019) fused to either sfGFP or mEGFP for intracellular visualization.…”

“…The reverse approach, that is, move or trap the “single-tagged” POI with an anchored binder, is a possible functionalization of these small tag binders. Mislocalization or trapping of some POIs, tagged with FP, has been developed with anti-GFP nanobodies (Harmansa et al, 2017; Seller et al, 2019), anti-mTFP DARPin (Vigano et al, 2018), anti-mCherry nanobody (Prole and Taylor, 2019) but also with nanobodies against endogenous, non-tagged proteins, for example Gelsolin and CapG (Van Audenhove et al, 2013).…”

“…Ideally, and to minimize the potential perturbation of the POI, the latter could be tagged by a short peptide to which high affinity protein binders have been identified and characterized; this approach would allow the use of characterized and validated binders and results in minimal potential disturbance of the function of the POI. Multiple protein manipulation tools generated with nanobodies or DARPins directed towards FPs (Aguilar et al, 2019a; Beghein and Gettemans, 2017; Brauchle et al, 2014; Schumacher et al, 2018; Vigano et al, 2018) could be adapted in order to functionalize small tag binders.…”

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

“…The reverse approach, that is, move or trap the single-tagged POI with an anchored binder, is a possible functionalization of these small tag binders. Mislocalization or trapping of some POIs, tagged with FP, has been developed with anti-GFP nanobodies (Harmansa et al, 2017;Seller et al, 2019), anti-mTFP DARPin (Vigano et al, 2018), anti-mCherry nanobody (Prole and Taylor, 2019) but also with nanobodies against endogenous, non-tagged proteins, for example Gelsolin and CapG (Van Audenhove et al, 2013). Moreover, a very recent report showed the efficient trapping of an extracellular POI (Dpp), endogenously tagged with a single HA copy, using a functionalized anti-HA_scFv (Matsuda et al, 2020).…”

“…Ideally, and to minimize the potential perturbation of the POI, the latter could be tagged by a short peptide to which high-affinity protein binders have been identified and characterized; this approach would allow the use of available, well-characterized and validated binders, and results in minimal potential disturbance of the function of the POI. Multiple protein manipulation tools generated with nanobodies or DARPins directed towards FPs ( Aguilar et al, 2019a ; Beghein and Gettemans, 2017 ; Brauchle et al, 2014 ; Schumacher et al, 2018 ; Vigano et al, 2018 ) could be adapted in order to functionalize small tag binders.…”