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 and Richard ŠTEFL

Basic information

Original name

Efficient and robust preparation of tyrosine phosphorylated intrinsically disordered proteins

Authors

BRÁZDA, Pavel (203 Czech Republic, belonging to the institution), Ondrej ŠEDO (203 Czech Republic, belonging to the institution), Karel KUBÍČEK (203 Czech Republic, belonging to the institution) and Richard ŠTEFL (203 Czech Republic, guarantor, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 1.541

RIV identification code

RIV/00216224:14740/19:00107820

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.2144/btn-2019-0033

UT WoS

000475967900005

Keywords in English

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

Abstract

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.

Links

GA15-17670S, research and development project

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

Investor: Czech Science Foundation

GA18-11397S, research and development project

Name: Molekulární achitektura komplexů ADAR - RNA

Investor: Czech Science Foundation

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

649030, interní kód MU

Name: DECOR - Dynamic assembly and exchange of RNA polymerase II CTD factors (Acronym: DECOR)

Investor: European Union, DECOR, ERC (Excellent Science)

90043, large research infrastructures

Name: CIISB

Files attached

btn-2019-0033.pdf

Find similar documents

Citovat

BRÁZDA, Pavel, Ondrej ŠEDO, Karel KUBÍČEK and 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, vol. 67, No 1, p. 16-22. ISSN 0736-6205. Available from: https://dx.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 = {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},
   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 and 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, vol.~67, No~1, p.~16-22. ISSN~0736-6205. Available from: https://dx.doi.org/10.2144/btn-2019-0033.

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