Nanobodies have biochemical properties that favour a wide variety of biological applications: they are small in size, they are highly stable, and they do not, in many cases, require disulphide bridges to correctly fold – in such cases, nanobodies can be stably expressed within the cytosol as intrabodies ( Dmitriev et al., 2016 Kaiser et al., 2014). It was discovered that the antigen-binding domain of HcABs can be reduced to a single variable domain (V HH, also called a nanobody) of only ∼12-15 kDa ( Fig. 1C,D). The discovery of naturally evolved heavy chain antibodies (HcABs) in camelids ( Hamers-Casterman et al., 1993) initiated a new era in antibody engineering ( Beghein and Gettemans, 2017 Goldman et al., 2017). These will no doubt start to play an increasingly important role in intracellular protein targeting, especially as scFvs can be derived from the huge pool of existing and validated monoclonal antibodies and used as genetically encoded protein binders in cells and developing organisms. Nonetheless, using protein engineering ( Tanha et al., 2006) and specific screening strategies, several improved scFv binders have been successfully generated ( Lynch et al., 2008 Vielemeyer et al., 2010) and scFvs can now be selected for intracellular use (e.g. Owing to these limitations, relatively few scFv-based binders have been used to date in developmental biology. These bonds influence scFv stability and function ( Glockshuber et al., 1992 Proba et al., 1997), and so only intrinsically stable scFvs fold correctly within a cell and can be utilized as functional intrabodies ( Worn and Pluckthun, 1998). These advantages make scFvs attractive tools in medical applications and in biotechnology ( Lyon and Stasevich, 2017 Monnier et al., 2013) but their use as intracellular protein binders (intrabodies) is restricted because scFvs typically contain two highly conserved intra-domain disulphide bonds ( Williams and Barclay, 1988). scFvs are relatively small (∼28 kDa) and consist of a single domain that can be expressed in various host systems, such as bacteria, yeast or higher animals. These drawbacks are partially overcome by connecting the V H and V L domain with a peptide linker, forming a so-called single-chain variable fragment (scFv Bird et al., 1988 Huston et al., 1988 see Fig. 1B) that retains antigen-binding capacity. 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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