RT Journal Article
SR Electronic
T1 DARPins recognizing mTFP1 as novel reagents for <em>in vitro</em> and <em>in vivo</em> protein manipulations
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 354134
DO 10.1101/354134
A1 M. Alessandra Vigano
A1 Dimitri Bieli
A1 Jonas V. Schaefer
A1 Roman Peter Jakob
A1 Shinya Matsuda
A1 Timm Maier
A1 Andreas Plückthun
A1 Markus Affolter
YR 2018
UL http://biorxiv.org/content/early/2018/06/22/354134.abstract
AB Over 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.Summary statement Structural 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.