Antibodies (immunoglobulins) have widely served as biochemical tools for molecular recognition in biological research and medicine for more than a century. Currently, they even constitute an accepted class of biopharmaceuticals with a huge market volume. The recent success of humanized or fully human monoclonal antibodies as therapeutic agents is a result of their high target specificity, therapeutic efficacy, safety and, consequently, low failure rate during preclinical and clinical development. However, immunoglobulins are not the only class of proteins that can be employed for the tight and specific binding of target molecules, a task necessary for inhibition, detection and separation purposes in biomedical research, biotechnology and clinical therapy. Alternative protein scaffolds that permit the display of sets of engineered peptide loops or side-chain arrays to yield novel binding sites have attracted increasing attention. A common feature is that they exhibit a structurally rigid core, which can present surface loops of varying sequence and length or tolerate diverse side-chain combinations in a contiguous surface region. In principle, binding proteins with prescribed target recognition properties can be obtained either by rational grafting of peptide loops with known binding activities or via site-directed random mutagenesis of exposed side chains in combination with molecular selection techniques (e.g. phage display). Based on the robust nature of the underlying scaffold, the resulting proteins should retain beneficial properties, such as high expression yield in microbial host organisms, stability against thermal or chemical denaturation, proteolytic resistance and suitability for functional fusion with reporter enzymes and the like. This minireview series covers three representative examples from this group: the affibodies (by P.-Å. Nygren), the anticalins (by A. Skerra) and the cystine-knot miniproteins (by H. Kolmar). These classes of established protein scaffolds have distinct structural features. Affibodies rely on a three-helix bundle supersecondary structure derived from bacterial protein A, which provides a rigid framework for displaying diverse side-chain arrays on one side. Specificities for many different proteins, such as Taq DNA polymerase and the ErbB2 receptor, have been obtained using this approach. Anticalins are constructed on the basis of a stable β-barrel fold that is characteristic for the lipocalin family of proteins. By variation of the four peptide–loop segments that form the entrance to the natural binding site, novel specificities both for small molecule ligands (e.g. digoxigenin) and for large protein targets (e.g. cytotoxic T-lymphocyte antigen-4) have been achieved. Cystine-knot miniproteins owe their high chemical stability to a central arrangement of three disulphide bridges that connect several loop segments. This scaffold is nicely suited for the grafting of peptides with defined biochemical activities (e.g. from thrombopoietin), conferring increased stability against degradation. These examples illustrate the enormous potential for the engineering of binding proteins outside the immunoglobulin field, thus providing reagents with favourable properties for diverse applications. At present, the generation of novel biopharmaceuticals based on alternative scaffolds raises great commercial expectations. Moreover, tailored binding proteins should also be of value in basic biological research as well as in biotechnology and nanotechnology, thus facilitating new experimental approaches.
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Kolmar et al. (2008) studied this question.