Espressione eterogenea di Bestrhodopsin da Cymbomonas tetramitiformis e la sua analisi funzionale

Rhodopsins are unique G-protein coupled receptors working as photoreceptor proteins, abundant in all domains of life. They can function differently to give rise to physiological response, transduce the information or react to a signal. On the other hand, bestrophin is a ligand-gated ion channel mainly found in metazoans including humans. Bestrhodopsin is a unique subfamily of microbial rhodopsin consisting of both rhodopsin and bestrophin. It is a light-activated large pentameric channel with a molecular weight of 700kD, which consists of one or two tandemly placed rhodopsins forming pseudo-dimers and surrounding a bestrophin channel in the middle. The analysis of the protein structure revealed unique features, like the eight transmembrane domain architecture of rhodopsins instead of the seven transmembrane domain structures found in most microbial rhodopsins. The functional aspects are also exceptional, showing the retinal isomerizes from all-trans to 11-cis retinal upon illumination, instead of conventional all-trans to 13-cis retinal inìsomerization found in other microbial rhodopsins. This is the first instance where the ion conduction pathway is not through the rhodopsin domain but through the bestrophin channel. The analysis of the genomic DNA library of the TARA oceans program first revealed the existence of such a protein. Phylogenetic analysis of the sequence showed the existence of this protein in other organisms, especially in dinoflagellates. Cymbomonas tetramitiformis is a green algae found in oceans, the previous study predicted that they have the gene to code the protein. In this project, we tried to express this protein for further structural and functional analysis. Different molecular cloning strategies were used to prepare vectors for transformation in different heterologous systems, followed by expression and purification of the protein. The expression was optimised and the purified protein was characterised through SDS-PAGE and absorption spectral analysis. Our analysis revealed that the deprotonated form of the protein was expressed and further studies are required to get its fully functional protonated form.