Technical perspective of investigating proteins involved in the plant
hormone system
Klimeš P.1,2
, Turek D.1
, Mazura P.1
, Spíchal L.3
, Gallová L.3
, Brzobohatý B.1
1. Laboratory of Plant Molecular Biology, Institute of Biophysics AS CR, v.v.i. and CEITEC – Central European
Institute of Technology, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic
2. Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
3. Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and
Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech
Republic
This study is mainly focused on two proteins involved in plant hormone cytokinin
metabolism and signalling, β-D-glucosidase and CRE1/AHK4 receptor.
β-D-glucosidase releases physiologically active cytokinins from their O-glucosides.
We used mutagenesis to study structural relationships determining β-D-glucosidase specificity.
The saturation mutagenesis was performed by combination of random saturation mutagenesis
and site-direct mutagenesis. Mutants were expressed in E. coli and purified using two-step
purification process (affinity chromatography and gel filtration). The initial velocities using
one concentration of the natural as well as artificial substrate were determined.
β-D-glucosidase was also used as a model enzyme to develop an automated method for
enzyme kinetics using artificial as well as natural substrates. For the automation using natural
substrate was used the previously developed method that was based on glucose detection.
These methods were optimized using liquid handling system BioNex Nanodrop II.
The cytokinin receptor CRE1/AHK4 transduces the hormone signal via interaction with
its agonist including compounds released by β-D-glucosidase. In this study bacteria E. coli
KMI001 transformed with plasmid carrying gene for CRE1/AHK4 was used. The modified
bacterial signaling pathway leads into expression of reporter gene coding β-D-galactosidase.
High throughput screening method based on this assay was optimized for seeking receptor
agonists and antagonist across huge library of compounds. The method also includes process
to identify compounds that interfere with the determination of the fluorescent product after
β-D-galactosidase enzymatic reaction.
Acknowledgments
We thank the SCALED BIOSYSTEMS team for help and useful ideas and suggestions. Access
to the MetaCentrum computing facilities provided under the program ‘‘Projects of Large
Infrastructure for Research, Development, and Innovations’’ LM2010005, funded by the
Ministry of Education, Youth, and Sports of the Czech Republic, is highly appreciated.