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

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Authors

BORKOVCOVÁ Petra ZOUHAR Jan HEJÁTKO Jan BRZOBOHATÝ Břetislav

Year of publication 2001
Type Article in Proceedings
Conference V. Pracovní setkání biochemiků a molekulárních biologů, Sborník příspěvků, Brno 2001
MU Faculty or unit

Faculty of Informatics

Citation
Field Genetics and molecular biology
Description 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.
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