However, conventional IgGs are unsuitable for intracellular expression for various reasons the reducing nature of the intracellular environment hampers disulphide bond formation and thus proper antibody folding, and whole IgG antibodies have a complex structure and a high atomic mass (∼150 kDa). Furthermore, such protein binders can be ‘functionalized’ by fusing them to various effector domains with the ultimate goal of directly visualizing or regulating the function and interaction of target proteins in living cells or organisms.Ĭonventional antibodies (immunoglobulins, IgGs see Fig. 1A) have been used extensively in basic research and are indispensable tools for protein detection. These genetically encodable binders, which are based on various protein scaffolds, can be used to block or mask protein function. This approach utilizes protein binders, which are small, protein-based affinity reagents that can selectively recognize and bind to a target protein and that are increasingly being used to study protein function in living cells and organisms ( Beghein and Gettemans, 2017 Helma et al., 2015 Plückthun, 2015 Sha et al., 2017). However, an additional, more systematic approach to manipulation of protein function has recently emerged. These include degradation-inducing applications ( Banaszynski et al., 2006 Bonger et al., 2011 Chung et al., 2015 Natsume et al., 2016), protein cleavage using tobacco etch virus (TEV) protease ( Harder et al., 2008 Pauli et al., 2008), the ‘anchor-away’ approach ( Haruki et al., 2008), the ‘knocksideways’ technique ( Robinson et al., 2010) and various dimerization tools that allow protein functions to be assembled in an inducible manner ( Renicke et al., 2013 van Bergeijk et al., 2015 Wu et al., 2009), to mention just a few. Over the years, several methods have been developed to manipulate proteins directly in vivo.
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