![]() ![]() Interest in small Ig binders was boosted by the discovery that camelids and cartilaginous fish have high-affinity single V-Ig that can be produced as soluble and stable in vitro products, and whose humanization makes them promising for clinical applications ( Nelson and Reichert, 2009).ĭifficulties with the production and intellectual property context of Ig-based binders triggered a search for alternative protein scaffolds. This problem can be alleviated by surface mutagenesis but success is variable ( Nelson and Reichert, 2009). Fragments, however, often suffer aggregation due to the hydrophobicity of their surface. These smaller binders also have functional advantages, being able to access cryptic epitopes and allowing the production of immunoconjugates by fusion to molecules such as enzymes for pro-drug therapy, toxins for cancer treatment and liposomes for drug delivery ( Holliger and Hudson, 2005 Nelson and Reichert, 2009). Antibody fragments and their engineered variants, that have simplified structures and can be optimized through design, are more amenable to economical bacterial expression. However, their complex architecture (requiring glycosylation and disulfide bond formation) makes them costly to manufacture, needing stably transfected mammalian cell lines ( Steinmeyer and McCormick, 2008). Thus, the V-Ig fold is regarded as the canonical protein scaffold.Īntibodies can be developed through immunization or in vitro display methods to exhibit high binding affinity and specificity. This Ig has three hypervariable loops (BC, C′C″, FG) at the N-terminal pole of the β-sandwich, which can accommodate large differences in sequence, length and conformation, being efficient display loci for binder motifs. Among these, the V(variable)-Ig type mediates antigen recognition in antibodies. Several fold variants are found in proteins that differ in β-strand composition and loop architecture, forming the Ig-like superfamily ( Halaby et al., 1999). The Ig fold (∼10 kDa) consists of two amphipathic β-sheets that pack against each other to form a β-sandwich. This versatility relies on the structural plasticity of the Ig fold of antibody domain components. Antibodies are naturally produced by the immune system through a process of hypermutation that yields myriads of variants with unique binding attributes. Fab, single-chain variable fragments (scFv), diabodies, triabodies and minibodies) that have been used as archetypal binders for decades ( Holliger and Hudson, 2005 Lo et al., 2008 Wurch et al., 2008 Nelson and Reichert, 2009). Many applications rely on antibodies, antibody fragments or their engineered variants (e.g. in medical imaging, immunoassays, microarrays and intracellular therapy. Protein scaffolds with adaptable molecular recognition properties are widely used in biotechnology and biomedicine, e.g. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin-in particular, modular components of the titin myofilament. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Protein scaffolds that support molecular recognition have multiple applications in biotechnology. ![]()
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