Efficient and robust preparation of tyrosine phosphorylated   
intrinsically disordered proteins

J 2019

Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins

BRÁZDA, Pavel; Ondrej ŠEDO; Karel KUBÍČEK a Richard ŠTEFL

Základní údaje

Originální název

Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins

Autoři

BRÁZDA, Pavel; Ondrej ŠEDO; Karel KUBÍČEK a Richard ŠTEFL

Vydání

BioTechniques, UNITED HOUSE, 2 ALBERT PL, LONDON N3 1QB, FUTURE SCI LTD, 2019, 0736-6205

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 1.541

Označené pro přenos do RIV

Ano

Kód RIV

RIV/00216224:14740/19:00107820

Organizační jednotka

Středoevropský technologický institut

Klíčová slova anglicky

C-terminal domain; co-expression; CTD; IDP; intrinsically disordered proteins; phosphorylation; purification; RNA polymerase II; TRANSCRIPTION TERMINATION; LARGEST SUBUNIT; CELL-CYCLE; KINASE; STRATEGIES; SEMISYNTHESIS

Štítky

CF NMR, CF PROT, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 14. 10. 2024 13:09, Ing. Jana Kuchtová

Anotace

V originále

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.

Návaznosti

GA15-17670S, projekt VaV

Název: Molekulární podstata pro specifické rozpoznávání histonu H3K4me2 pomocí Set3 PHD domény

Investor: Grantová agentura ČR, Molekulární podstata pro specifické rozpoznávání histonu H3K4me2 pomocí Set3 PHD domény

GA18-11397S, projekt VaV

Název: Molekulární achitektura komplexů ADAR - RNA

Investor: Grantová agentura ČR, Molecular architecture of ADAR - RNA complexes

LQ1601, projekt VaV

Název: CEITEC 2020 (Akronym: CEITEC2020)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, CEITEC 2020

649030, interní kód MU

Název: DECOR - Dynamic assembly and exchange of RNA polymerase II CTD factors (Akronym: DECOR)

Investor: Evropská unie, DECOR - Dynamic assembly and exchange of RNA polymerase II CTD factors, ERC (Excellent Science)

90043, velká výzkumná infrastruktura

Název: CIISB

Přiložené soubory

btn-2019-0033.pdf Verze souboru

Citovat

BRÁZDA, Pavel; Ondrej ŠEDO; Karel KUBÍČEK a Richard ŠTEFL. Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins. BioTechniques. UNITED HOUSE, 2 ALBERT PL, LONDON N3 1QB: FUTURE SCI LTD, 2019, roč. 67, č. 1, s. 16-22. ISSN 0736-6205. Dostupné z: https://doi.org/10.2144/btn-2019-0033.

@article{1585263,
   author = {Brázda, Pavel and Šedo, Ondrej and Kubíček, Karel and Štefl, Richard},
   article_location = {UNITED HOUSE, 2 ALBERT PL, LONDON N3 1QB},
   article_number = {1},
   doi = {https://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},
   language = {eng},
   issn = {0736-6205},
   journal = {BioTechniques},
   title = {Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins},
   url = {https://www.future-science.com/doi/full/10.2144/btn-2019-0033},
   volume = {67},
   year = {2019}
}

TY  - JOUR
ID  - 1585263
AU  - Brázda, Pavel - Šedo, Ondrej - Kubíček, Karel - Štefl, Richard
PY  - 2019
TI  - Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins
JF  - BioTechniques
VL  - 67
IS  - 1
SP  - 16-22
EP  - 16-22
PB  - FUTURE SCI LTD
SN  - 07366205
KW  - C-terminal domain
KW  - co-expression
KW  - CTD
KW  - IDP
KW  - intrinsically disordered proteins
KW  - phosphorylation
KW  - purification
KW  - RNA polymerase II
KW  - TRANSCRIPTION TERMINATION
KW  - LARGEST SUBUNIT
KW  - CELL-CYCLE
KW  - KINASE
KW  - STRATEGIES
KW  - SEMISYNTHESIS
UR  - https://www.future-science.com/doi/full/10.2144/btn-2019-0033
L2  - https://www.future-science.com/doi/full/10.2144/btn-2019-0033
N2  - 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.
ER  -

BRÁZDA, Pavel; Ondrej ŠEDO; Karel KUBÍČEK a Richard ŠTEFL. Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins. \textit{BioTechniques}. UNITED HOUSE, 2 ALBERT PL, LONDON N3 1QB: FUTURE SCI LTD, 2019, roč.~67, č.~1, s.~16-22. ISSN~0736-6205. Dostupné z: https://doi.org/10.2144/btn-2019-0033.

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