Institute of Medical Genetics, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Währinger Straße 10, A-1090, Vienna, Austria.
Vienna Doctoral School of Pharmaceutical, Nutritional and Sport Sciences (PhaNuSpo), University of Vienna, Althanstraße 14, A-1090, Vienna, Australia.
Protein J. 2022 Apr;41(2):345-359. doi: 10.1007/s10930-022-10054-9. Epub 2022 May 12.
The transcriptional regulator Methyl-CpG-binding protein 2 (MeCP2) is an intrinsically disordered protein, mutations in which, are implicated in the onset of Rett Syndrome, a severe and debilitating neurodevelopmental disorder. Delivery of this protein fused to the cell-penetrating peptide TAT could allow for the intracellular replenishment of functional MeCP2 and hence potentially serve as a prospective Rett Syndrome therapy. This work outlines the expression, purification and characterization of various TAT-MeCP2 constructs as well as their full-length and shortened eGFP fusion variants. The latter two constructs were used for intracellular uptake studies with subsequent analysis via western blotting and live-cell imaging. All purified MeCP2 samples exhibited high degree of stability and very little aggregation propensity. Full length and minimal TAT-MeCP2-eGFP were found to efficiently transduce into human dermal and murine fibroblasts and localize to cell nuclei. These findings clearly support the utility of MeCP2-based protein replacement therapy as a potential Rett Syndrome treatment option.
转录调节因子甲基化CpG 结合蛋白 2（MeCP2）是一种固有无序的蛋白质，其突变与雷特综合征（一种严重的神经发育障碍）的发病有关。将与穿膜肽 TAT 融合的这种蛋白质递送到细胞内，可以补充有功能的 MeCP2，因此可能成为一种有前途的雷特综合征治疗方法。这项工作概述了各种 TAT-MeCP2 构建体以及它们的全长和缩短的 eGFP 融合变体的表达、纯化和特性。后两种构建体用于细胞内摄取研究，随后通过 Western blot 和活细胞成像进行分析。所有纯化的 MeCP2 样品都表现出高度的稳定性和非常低的聚集倾向。全长和最小 TAT-MeCP2-eGFP 被发现能够有效地转导到人真皮和成纤维细胞中，并定位于细胞核。这些发现清楚地支持基于 MeCP2 的蛋白质替代治疗作为一种潜在的雷特综合征治疗选择的实用性。
