Indeed, unregulated activity has been implicated in the pathogenesis of many diseases (e.g. Because unregulated proteolysis in any of these processes will have undesirable effects, effective control of protease activity is critical. Proteases have important biological functions, they have been identified as key regulators of cellular processes such as ovulation, fertilization, wound healing, angiogenesis, apoptosis, peptide hormone release, coagulation and complement activation (Nixon and Wood, 2006). The smaller antigen-binding antibody fragments are shown, that is, Fab, scFv, and the single-domain antibody (sdAb) fragment, VH.Īs therapeutics, globular proteins with engineered binding properties might be particularly interesting, (i) if the neutralization of a target protein is the desired pharmacological effect (in contrast to a full length antibody, where the Fc portion may stimulate immune processes), (ii) as fusion proteins, for the targeted delivery of bioactive molecules to sites of disease, (iii) as receptor-binding drugs, thus interfering with the cell-signalling and (iv) as enzyme inhibitors.Ĭommon to all approaches of finding a suitable scaffold are the following steps (Nygren and Uhlen, 1997 Smith 1998 Binz and Pluckthun, 2005): The light chain is composed of one variable (VL) and one constant (CL) domain, whereas the heavy chain has one variable (VH) and three constant domains (CH1, CH2, and CH3). Classical IgG consists of two light chains and two heavy chains. H, Schematic representation of full-sized antibodies, multidomain and single-domain antigen-binding fragments ( Saerens et al., 2008). The three disulfide bonds of RBP are depicted in yellow. The typical α-helix that is attached to the central β-barrel in all lipocalins, the loops at the closed end the N- and C-terminal peptide segments are shown in grey. The eight antiparallel strands of the conserved β-barrel structure are shown in blue with labels A to H, and the four loops, which are highly variable among the lipocalin family, are colored red and numbered. Ribbon diagram of the crystal structure of RBP with the bound ligand retinol (magenta, ball and stick representation). G, General structure of human retinol-binding protein, a prototypic lipocalin ( Schlehuber and Skerra, 2005). (lower part) Ribbon representation of the selected MBP binding ankyrin repeat protein (colours as above) This binder was isolated from a library of N-terminal capping ankyrin repeat, three designed ankyrin repeat modules and a C-terminal capping ankyrin repeat. (upper part) Combinatorial libraries of ankyrin repeat proteins were designed by assembling an N-terminal capping ankyrin (green), varying numbers of the designed ankyrin repeat module (blue) and a C-terminal capping ankyrin (cyan) side chains of the randomized residues are shown in red. F, Schematic representation of the library generation of designed ankyrin repeat proteins ( Binz et al., 2004). For clarity, only the electron density around the side chains is displayed. E, Electron density for all 13 mutated residues in the affibody ( Hogbom et al., 2003). D, Fyn SH3 wt protein structure (Protein Data Bank entry 1M27) The RT-Src loop is in red, and the n-Src loop is in green. A ribbon diagram of a prototypical A-domain structure is included (Protein Data Bank entry 1AJJ). Calcium-coordinating scaffold residues are yellow, structural scaffold residues are blue, cysteine residues are red and variable positions are green. Each circle represents an amino acid position. C, Fixed and variable positions of the A-domain library, as well as the disulfide topology, are indicated ( Silverman et al., 2005). The CDRs of the V HH domain and the residues randomized in the 10Fn3 domain are shown in color. Despite the lack of significant sequence identity, both domains fold into similar beta sheet sandwiches. B, Structural comparison of a llama V HH domain and the wild-type human 10Fn3 domain ( Xu et al., 2002). Varied positions are depicted in black, the P1 and second loop positions are enclosed. A, Kunitz type domain (LAC–D1) (Ley et al., 1996). 13.1 Schematic representation of representative scaffolds.
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