Nakada-Masuta, T.; Takeda, H.; Uchida, K. Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica). Mar. Drugs2023, 21, 550.
Nakada-Masuta, T.; Takeda, H.; Uchida, K. Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica). Mar. Drugs 2023, 21, 550.
Nakada-Masuta, T.; Takeda, H.; Uchida, K. Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica). Mar. Drugs2023, 21, 550.
Nakada-Masuta, T.; Takeda, H.; Uchida, K. Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica). Mar. Drugs 2023, 21, 550.
Abstract
Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. We constructed a VNAR phage library from the spleen of the Japanese topeshark (Hemitriakis japanica). Drawing inspiration from the physiological conditions of sharks and physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. NGS analysis was performed to select the specifically enriched clones under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.
Keywords
Japanese topeshark; VNAR; phage display; NGS; selection pressures
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
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