| 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. |
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@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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