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
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.