Abstract
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, several affinity reagents against fluorescent proteins have also 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 fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.
Highlights
Over recent decades, much has been learned about the role of different proteins in controlling cell proliferation, cell movement, cell determination and cell differentiation, both in cell culture and during the development of multicellular animals
Selection and in vitro characterization of monomeric teal fluorescent protein 1 (mTFP1)-binding designed ankyrin repeat proteins (DARPins) To generate suitable DARPin binders, streptavidin-binding peptide (SBP)-tagged mTFP1 was immobilized on streptavidin beads and used as a target for DARPin selections by employing multiple rounds of Ribosome Display (Dreier and Plückthun, 2012; Plückthun, 2012)
400 colonies of transformed E. coli were picked and the encoded DARPins were expressed in small scale
Summary
Much has been learned about the role of different proteins in controlling cell proliferation, cell movement, cell determination and cell differentiation, both in cell culture and during the development of multicellular animals. Received 22 June 2018; Accepted 10 September 2018 screens (Brenner, 1974; Nüsslein-Volhard and Wieschaus, 1980) Later, these approaches were complemented with reverse genetic approaches, which allowed for loss- and gain-of-function studies that could be controlled with regard to developmental time and to the targeted tissue (Anderson et al, 2017; Housden et al, 2017; Nagarkar-Jaiswal et al, 2015, 2017; Venken et al, 2011; Yamamoto et al, 2014). While off-target effects have to be taken into account when studying protein function with these methods, these approaches, in particular RNAi, allow for time- and tissue-controlled, genome-wide loss-offunction analyses (Housden et al, 2017). When studying proteins with a long half-life, or in cases of maternal contribution of proteins in early embryos, it might be very difficult or even impossible to deplete the protein of interest and analyse its contribution to cellular or organismal function
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