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Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins
P. Brázda, O. Šedo, K. Kubíček, R. Štefl,
Language English Country Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
NLK
Directory of Open Access Journals
from 2018
Freely Accessible Science Journals
from 1996
Taylor & Francis Open Access
from 1996-01-01
ROAD: Directory of Open Access Scholarly Resources
from 1983
PubMed
31092000
DOI
10.2144/btn-2019-0033
Knihovny.cz E-resources
- MeSH
- Escherichia coli genetics MeSH
- Gene Expression MeSH
- Phosphorylation MeSH
- Humans MeSH
- Nuclear Magnetic Resonance, Biomolecular MeSH
- Protein Processing, Post-Translational MeSH
- Protein Domains MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Transformation, Genetic MeSH
- Tyrosine analysis genetics MeSH
- Intrinsically Disordered Proteins chemistry genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system: RNA polymerase II C-terminal domain (CTD); 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 the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.
References provided by Crossref.org
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