Department of Environmental Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China.
Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China.
Biotechnol Lett. 2022 Apr;44(4):613-621. doi: 10.1007/s10529-022-03246-x. Epub 2022 Mar 31.
To study the effect of SpyTag/SpyCatcher cyclization on stability and refolding of protein, we constructed a cyclized green fluorescent protein (SRGFP) and its derivative to act as a linear structure control (L-SRGFP). SRGFP and L-SRGFP showed similar fluorescence characteristics to the wild-type GFP, while compared with GFP and L-SRGFP, the thermal stability and denaturation resistance of SRGFP were improved. The refolding efficiencies of these three denatured proteins were investigated under different pH, temperature and initial protein concentration conditions, and it was found that SRGFP was superior to GFP and L-SRGFP in terms of refolding yield and refolding speed. In the pH range of 8.0-8.5, SRGFP could basically recover all fluorescence, while GFP and L-SRGFP recovered only about 87.52% and 88.58%. When refolded at a high temperature (37 °C), SRGFP still recovered 85.27% of the fluorescence, whereas GFP and L-SRGFP recovered only around 69.43% and 68.45%. At a high initial protein concentration (5 mg/mL), the refolding yield of SRGFP was about 15% higher than that of both GFP and L-SRGFP. These results suggest that the introduction of SpyRing structure (head-to-tail cyclization via SpyTag and SpyCatcher) improved the protein's stability and facilitated the refolding of denatured protein.
为了研究 SpyTag/SpyCatcher 环化对蛋白质稳定性和复性的影响，我们构建了环化的绿色荧光蛋白（SRGFP）及其衍生物作为线性结构对照（L-SRGFP）。SRGFP 和 L-SRGFP 表现出与野生型 GFP 相似的荧光特性，而与 GFP 和 L-SRGFP 相比，SRGFP 的热稳定性和抗变性能力得到了提高。在不同 pH、温度和初始蛋白浓度条件下，研究了这三种变性蛋白的复性效率，发现 SRGFP 在复性产率和复性速度方面均优于 GFP 和 L-SRGFP。在 pH 为 8.0-8.5 的范围内，SRGFP 基本上可以恢复所有荧光，而 GFP 和 L-SRGFP 仅恢复了约 87.52%和 88.58%。在高温（37°C）下复性时，SRGFP 仍能恢复 85.27%的荧光，而 GFP 和 L-SRGFP 仅恢复约 69.43%和 68.45%。在初始蛋白浓度较高（5mg/mL）时，SRGFP 的复性产率比 GFP 和 L-SRGFP 分别高出约 15%。这些结果表明，引入 SpyRing 结构（通过 SpyTag 和 SpyCatcher 头尾环化）提高了蛋白质的稳定性，并促进了变性蛋白的复性。
