Akademik Veri Yönetim Sistemi
INSAC International Researches Congress on Natural and Engineering Sciences (INSAC-IRNES'23), Konya, Türkiye, 18 - 19 Mart 2023, ss.203-204
Being located at the interface of intracellular and extracellular regions of biological cells, membrane proteins (MPs) mediate many vital events including signal transduction, energy generation, selective transport of solutes etc.. Because of these regulatory properties, nearly 70% of currently FDA approved drugs on the market target membrane proteins. Despite their importance, low abundance of MPs in natural resources is one of the major bottlenecks in studying their structure and functionality. Moreover, MPs have been employed in miscellaneous applications such as sensor technology, optogenetics, biomimetic separation membranes, in-vitro photosynthesis leads to an ever increasing demand for avaliability of MPs. Recombinant expression of MPs from heterologous expresion hosts was proven to be challenging especially for mammalian MPs due to improper folding, inclusion body formation and toxic effects on host organism. Therefore, development of alternative expression host systems for functional MP expression is of critical importance. Purple non-sulphur bacteria from Rhodobacter genus are known for their diverse metabolic pathways and intensively studied as model organisms in photosynthetis research. Under anaerobic, photosynthetic growth mode, plasma membrane surface area of Rhodobacter ssp immensely increase to accomodate the photosynthetic apparatus. An order of magnitude higher membrane surface area with inducible expression vectors imparts Rhodobacter sp as an attractive recombinant MP expression host. In this study, we employed codon optimized MISTIC chaperonin protein gene as N-terminal and mBanana fluorescent protein gene as C-terminal fusion partner for human AQP6 and AQP9 genes for co-expression in Rhodobacter sp. Cloning procedures were carried out in E. coli Top10 and expression vectors were transfered in Rhodobacter by biparental mating using E. coli S17 -pir. Protein expression was investigated under semi-aerobic and photosynthetic growth modes using two different promoters. A significant improvement in expression titers was observed with MISTIC fusions as monitored by SDS-PAGE.