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. 2021 Jul 26;8:686086. doi: 10.3389/fmolb.2021.686086

FIGURE 1.

FIGURE 1

Chemical and enzymatic methods for the generation of Httex1-43Q-oxM8, Httex1-43Q-oxM8/pT3, and Httex1-43Q-oM8/AcK6. (A) Schematic presentation of the Httex1 sequence, highlighting the cluster of PTMs in the Nt17 domain. (B) Schematic presentation of the SUMO-based strategy used to produce Httex1-43Q-oxM8 and Httex1-43Q-oxM8/pT3. The SUMO-Httex1-43Q was produced and purified by Nickel IMAC purification and subsequently oxidized or both oxidized and phosphorylated by GCK. Next, the SUMO tag was removed by ULP1, and the desired protein was purified by RP-HPLC. (C) Schematic representation for the semisynthetic strategy used for the generation of mHttex1-oxM8/AcK6 (adapted from Chiki et al. (2017)). (D) Characterization of Httex1-43Q-oxM8, Httex1-43Q-oxM8/pT3, Httex1-43Q-oM8/AcK6, and unmodified Httex1-43Q by ESI/MS, UPLC, and SDS-PAGE.