Structural study on C-terminus of the vanilloid receptor TRPV1
V. Mráziková1, E. Jindrová1, V. Vlachová2 , R. Ettrich1, J. Teisinger2
1Laboratory of High Performance Computing, Institute of Systems Biology and Ecology ASCR
Institute of Physical Biology USB Zámek 136, CZ-373 33 Nové Hrady,
2Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Praha 4
The vanilloid receptor (TRPV1) is a member of TRP channel family and has function as a multimodal signal transducter of noxious stimuli in the mammalian somatosensory system . The TRPV1 is consisted of six transmembarane-spanning domains with a pore forming region between fifth and sixth domains, and cytoplasmically located C – and N – terminal regions. Although structural and functional studies have been done [2, 3], the possible contributions of terminal regions to vanilloid receptor function remain elusive. To determine structure and functional properties of the cytoplasmically located tail, the DNA fragment encoding the C- terminus was cloned to the expression vector pGEX – 4T – 1 and transformed to E. coli strain (BL 21 (DE3)). Protein was expressed as fusion protein with the 26 kDa glutathione S-transferase (GST) and was purified by affinity and ion-exchange chromatography. Because of the denaturation of protein and very weak expression output we decided for subcloning to another expression vector pET 32a (+). The pET system is developed for expression of difficult soluble protein. Target genes were cloned in the pET 32a (+) under control of strong bacteriophage T7 transcription , in fusion with genes encoded His – tag S – tag hook and purified by using affinity chroamtography. Purified protein will be used for measuring by vibrational spectroscopy. Experimental results will be combined with homology and energetic modeling techniques and we will propose a three – dimensional structure of the C – terminus.
This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (MSM6007665808, LC06010) and by the Academy of Sciences of the Czech Republic (Institutional research concept AVOZ60870520)/
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