criteria for classification as a protein scaffold

The resulting combinatorial library serves as a source of proteins specific to a target of choice for in vitro display technologies, which give rise to ABPs possessing antibody-like specificity and selectivity to the target. An APS possesses a compact stable backbone supporting the target-binding regions, which are genetically randomized to provide a wide repertoire (10 5−10 13) of variants with retained structural stability.

Antibody alternatives, such as small antibody mimetics, alternative binding proteins (ABPs), based on immunoglobulin-like or non-immunoglobulin folds (‘alternative protein scaffolds’, APSs) have the potential to address these shortcomings. Though antibodies are traditionally used for these purposes, they suffer from several fundamental disadvantages related to their complex architecture (multi-subunit structure and abundance of post-translational modifications), including limited tissue penetration and access to antigen grooves, need for use of expensive eukaryotic expression systems, and the complicated process of their structural characterization. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.ĭevelopment of proteins capable of specific recognition of biological targets has numerous applications in biotechnology, diagnostics, therapy and research. provided support in the form of salary for author TAM. provided support in the form of salary for author TAM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: Antherix and Biomirex Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by a grant from the Program of the Russian Academy of Sciences «Fundamental science for medicine» (SEP) and grant №14-44-03604 from the Russian Foundation for Basic Research and the Ministry of Investments and Innovations of the Moscow region (SEP). Received: SeptemAccepted: JanuPublished: January 19, 2017Ĭopyright: © 2017 Lomonosova et al. PLoS ONE 12(1):Įditor: Zhaozhong Han, Alexion Pharmaceuticals Inc, UNITED STATES

Hence, 元5Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.Ĭitation: Lomonosova AV, Ulitin AB, Kazakov AS, Mirzabekov TA, Permyakov EA, Permyakov SE (2017) Derivative of Extremophilic 50S Ribosomal Protein 元5Ae as an Alternative Protein Scaffold. Overall, the CDR-like loop regions of 元5Ae 10X represent a proper interface for generation of functional ABPs. Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization.

L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference 元5Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). The reference 元5Ae 10X is non-specific to both HEL and BLA. While L4 has 1–2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data.

Two 元5Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. A phage display library of 元5Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×10 8). Here, a 10X mutant of the 50S ribosomal protein 元5Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics.

Criteria for classification as a protein scaffold