Preparation of AHP proteins for structural
analysis
Radka Dopitová1,2, Eliška
Nejedlá1,2, Oksana Degtjarik3,4, Martina Válková1,2,
Ivana Kutá-Smatanová3,4, Jan Hejátko1,2 and Lubomír Janda1,2
1Department
of Functional Genomics and Proteomics,
2Central
European Institute of Technology, Masaryk University, Žerotínovo nám. 9,
CZ-60177 Brno, Czech Republic
3Institute of Physical Biology,
University of South Bohemia, České Budějovice, Zámek 136, 373 33 Nové Hrady,
Czech Republic
4Institute of Systems
Biology and Ecology, Academy of Science of the Czech Republic, Zámek 136, 373
33 Nové Hrady, Czech Republic
A histidine-containing phosphotransmitters
(HPts) from A. thaliana (AHP1-5)
mediate signal transduction downstream from receptor histidine kinases (HK) to
subsequent phospho-accepting response regulators (RR) via so called multistep
phosphorelay (MSP). AHP proteins are involved in and potentially integrate various
MSP signalling pathways (e.g. cytokinin, ethylene, osmosensing). In this study,
we cloned genes for AHP proteins into pRSET B expression vector enabling production
of AHPs in fusion with N- terminal His tag. Expression levels were tested at
different cultivation conditions in BL21(DE3)pLysS E.coli host strain. Proteins were extracted under denaturing and
native conditions to estimate the proportion of produced AHPs in the soluble
and insoluble form, respectively. Both forms of respective protein were
identified by Western blotting. Surprisingly, quantification of bands from Western
blot analysis showed significant differences in expression levels of AHP
proteins. The first purification step (affinity chromatography) was optimized using
different buffers and gradient shape. Following purification step (gel
filtration or anion exchange chromatography) allowed obtaining of homogenous
proteins under denaturing condition. Purified AHP2 was used for crystallization
screening.
Supported by LC06034, LC06010, MSM0021622415,
GAČR 521/09/1699 and P305/11/0756.