Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
Molecules. 2017 Oct 10;22(10):1695. doi: 10.3390/molecules22101695.
Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.
与单克隆抗体相比，单链可变片段（scFv）的生产成本更低，因此具有在多种应用中的潜力，例如用于诊断和治疗一系列疾病，以及作为传感器元件。然而，scFv 的用途受到通过形成二聚体、三聚体和更大的寡聚体而导致的不均一性的限制。scFv 蛋白被假定处于 VH 和 VL 结构域组装或解组装形成的关闭和开放状态之间的平衡。因此，产生平衡偏向于关闭状态的 scFv 对于克服 scFv 在工业或治疗应用中的不均一性问题至关重要。在这项研究中，我们通过噬菌体展示系统和随机诱变的组合，获得了对 GA-吡啶（一种晚期糖基化终产物（AGE））稳定的 scFv 克隆。在 37°C 下进行生物淘选显著提高了 scFv 的稳定性。我们进一步通过小角 X 射线散射（SAXS）评估了回转半径，获得了紧凑的克隆体，并可视化了开放状态。
