Expression and Single-Step Purification of the Signal Receiver
Domain of  CKI1, a Putative Cytokinin Receptor from Arabidopsis
Thaliana.

D 2001

Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana.

BORKOVCOVÁ, Petra, Jan ZOUHAR, Jan HEJÁTKO a Břetislav BRZOBOHATÝ

Základní údaje

Originální název

Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana.

Autoři

BORKOVCOVÁ, Petra, Jan ZOUHAR, Jan HEJÁTKO a Břetislav BRZOBOHATÝ

Vydání

Brno, V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001, s. 30-30, 2001

Nakladatel

Masarykova univerzita v Brně

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

Genetika a molekulární biologie

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Kód RIV

RIV/00216224:14330/01:00004630

Organizační jednotka

Fakulta informatiky

ISBN

80-210-2538-7

Změněno: 23. 5. 2002 15:20, RNDr. JUDr. Vladimír Šmíd, CSc.

Anotace

V originále

Cytokinins are plant hormones involved in regulation of a number of responses in plants. Deciphering molecular mechanisms of cytokinin perception and signal transduction is a crucial step in analysis of molecular mechanism of cytokonin action. Recently, a putative cytokinin receptor, CKI1, has been identified in Arabidopsis thaliana by activation tagging. Sequence analysis revealed that CKI1 belongs to a family of sensor hybrid histidine kinases. We have subcloned individual CKI1 domains and investigated feasibility of their production in a bacterial expression system. Here we report a succesful production and single-step purification of the CKI1 signal receiver domain. The open reading frame coding for the domain was cloned into an expression vector pET-28 in a way leading to a fusion protein with two hexahistidine tags. The resulting fusion protein was produced in E. coli. Single-step purification of the fusion protein was achieved by immobilized metal affinity chromatography. Development of the purification scheme involved identification of the best performing combination of POROS-immobilized transition metal ion, and washing and elution conditions. The quantity and purity of the obtained recombinant protein was analyzed by SDS-PAGE and subsequent silver staining, Coomassie brillant blue staining and western blot analysis. We will report conditions yielding milligram quantities of the recombinant protein in purity higher than 95% from single run of a column.

Návaznosti

LN00A081, projekt VaV

Název: Signální dráhy u rostlin

VS96096, projekt VaV

Název: Laboratoř molekulární fyziologie rostlin

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Laboratoř molekulární fyziologie rostlin

Citovat

BORKOVCOVÁ, Petra, Jan ZOUHAR, Jan HEJÁTKO a Břetislav BRZOBOHATÝ. Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana. In V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001. Brno: Masarykova univerzita v Brně, 2001, s. 30. ISBN 80-210-2538-7.

@inproceedings{375034,
   author = {Borkovcová, Petra and Zouhar, Jan and Hejátko, Jan and Brzobohatý, Břetislav},
   address = {Brno},
   booktitle = {V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001},
   language = {eng},
   location = {Brno},
   isbn = {80-210-2538-7},
   pages = {30-30},
   publisher = {Masarykova univerzita v Brně},
   title = {Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana.},
   year = {2001}
}

TY  - JOUR
ID  - 375034
AU  - Borkovcová, Petra - Zouhar, Jan - Hejátko, Jan - Brzobohatý, Břetislav
PY  - 2001
TI  - Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana.
PB  - Masarykova univerzita v Brně
CY  - Brno
SN  - 8021025387
N2  - Cytokinins are plant hormones involved in regulation of a number of responses in plants. Deciphering molecular mechanisms of cytokinin perception and signal transduction is a crucial step in analysis of molecular mechanism of cytokonin action. Recently, a putative cytokinin receptor, CKI1, has been identified in Arabidopsis thaliana by activation tagging. Sequence analysis revealed that CKI1 belongs to a family of sensor hybrid histidine kinases. We have subcloned individual CKI1 domains and investigated feasibility of their production in a bacterial expression system. Here we report a succesful production and single-step purification of the CKI1 signal receiver domain. The open reading frame coding for the domain was cloned into an expression vector pET-28 in a way leading to a fusion protein with two hexahistidine tags. The resulting fusion protein was produced in E. coli. Single-step purification of the fusion protein was achieved by immobilized metal affinity chromatography. Development of the purification scheme involved identification of the best performing combination of POROS-immobilized transition metal ion, and washing and elution conditions. The quantity and purity of the obtained recombinant protein was analyzed by SDS-PAGE and subsequent silver staining, Coomassie brillant blue staining and western blot analysis. We will report conditions yielding milligram quantities of the recombinant protein in purity higher than 95% from single run of a column.
ER  -

BORKOVCOVÁ, Petra, Jan ZOUHAR, Jan HEJÁTKO a Břetislav BRZOBOHATÝ. Expression and Single-Step Purification of the Signal Receiver Domain of CKI1, a Putative Cytokinin Receptor from Arabidopsis Thaliana. In \textit{V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001}. Brno: Masarykova univerzita v Brně, 2001, s.~30. ISBN~80-210-2538-7.

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