Publication details
Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins
| Authors | |
|---|---|
| Year of publication | 2019 |
| Type | Article in Periodical |
| Magazine / Source | BioTechniques |
| MU Faculty or unit | |
| Citation | |
| Web | URL |
| Doi | http://dx.doi.org/10.2144/btn-2019-0033 |
| Keywords | C-terminal domain; co-expression; CTD; IDP; intrinsically disordered proteins; phosphorylation; purification; RNA polymerase II; TRANSCRIPTION TERMINATION; LARGEST SUBUNIT; CELL-CYCLE; KINASE; STRATEGIES; SEMISYNTHESIS |
| Attached files | |
| Description | Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to undertake different interaction networks with different consequences, ranging from regulatory signalling networks to formation of membraneless organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDP with subsequent purification procedure allowing production of modified IDP. The robustness of our protocol is demonstrated on a challenging system, RNA polymerase II C-terminal domain (CM), that is a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows to rapidly produce near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies. |
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