Abstract
Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. 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. 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 reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.
Highlights
Development of proteins capable of specific recognition of biological targets has numerous applications in biotechnology, diagnostics, therapy and research [1,2,3,4,5,6,7,8,9,10,11,12,13]
We have recently shown that 10X mutant (Fig 1A) of 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii [29] has several features favoring its use as a protein scaffold [30]: small size (88 residues), lack of disulfides, high thermal stability, efficient bacterial production (60 mg of protein per liter of cell culture), lack of non-specific binding to model human embryonic kidney 293 cells (HEK293), and the presence of three nearby loops closely resembling complementarity determining regions (CDRs) of immunoglobulins as judged from tertiary structure of L35Ae from P. furiosus (Fig 2A and 2C)
The residues of L35Ae 10X subjected to randomization form a double ‘paratopic’ region including two clusters of the residues provided by the CDR-like loop 1 and residues K74-G75 of loop 3, and loop 2 and residues G78-A80/D83 of loop 3, which are separated by residues L76-P77
Summary
Development of proteins capable of specific recognition of biological targets has numerous applications in biotechnology, diagnostics, therapy and research [1,2,3,4,5,6,7,8,9,10,11,12,13]. Though antibodies are traditionally used for these purposes [10,11,12], they suffer from several fundamental disadvantages related to their complex architecture L35Ae as an Alternative Protein Scaffold the manuscript. Antherix and Biomirex Inc. 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
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