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Neurofibrillary tangles composed of aggregated hyperphosphorylated Tau protein are one of the typical hallmarks of Alzheimer's disease (AD) in the brain [1]. As the patterns of phosphorylation and other post-translational modifications (PTMs) within Tau are complex [2], AD-relevant Tau protein is not easily obtainable by recombinant production in E. coli and then modification by individual enzymes in vitro. To overcome this problem, we employed mammalian cell culture (HEK293) to produce Tau protein with natural PTMs. The expression and purification conditions were optimized, and Tau protein purity and identity were verified by SDS-PAGE, Western blot, and mass spectrometry. Using LC-MS/MS, we identified around 20 phosphorylation sites with diverse extent of phosphorylation. The detected phosphorylation patterns were comparable to those found in the brains of AD-patients. Moreover, in contrast to Tau phosphorylated by protein kinase A (PKA) in vitro [3], Tau isolated from HEK293 and potentially AD-brains did not interact with 14-3-3? protein, a well-recognized Tau partner [4]. In the future, we plan to analyse also other properties of Tau purified from HEK293 cells – such as protein compactness, aggregation propensities and fibril folds – all in comparison to Tau produced in E. coli and phosphorylated by selected kinases in vitro [3]. The protein production in HEK293 cells was performed within an Industrial PhD programme in the biotech company BioVendor. Project acknowledgement: This project has received funding from the European Union’s Horizon Europe program under the grant agreement No. 101087124. We acknowledge CEITEC Proteomics Core Facility of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042, e-INFRA CZ (ID:90254)). References [1] M. Goedert, R. A. Crowther, S. H. W. Scheres, and M. G. Spillantini, “Tau and neurodegeneration,” Cytoskeleton, vol. 81, no. 1, pp. 95–102, 2024, doi: 10.1002/cm.21812. [2] H. Wesseling et al., “Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer’s Disease,” Cell, vol. 183, no. 6, pp. 1699-1713.e13, 2020, doi: 10.1016/j.cell.2020.10.029. [3] R. Crha et al., “Hiding in plain sight: Complex interaction patterns between Tau and 14-3-3? protein variants,” Int. J. Biol. Macromol., vol. 266, no. 130802, pp. 1–14, 2024, doi: 10.1016/j.ijbiomac.2024.130802. [4] J. F. Neves et al., “Phosphorylated full-length Tau interacts with 14-3-3 proteins via two short phosphorylated sequences, each occupying a binding groove of 14-3-3 dimer,” FEBS J., vol. 288, no. 6, pp. 1918–1934, 2021, doi: 10.1111/febs.15574.
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