Genetically Encoded FRET-Based Biosensor For Oxidation State Of The Cell

Oxygen is the main source of cells for energy, most of which is converted into water through the normal metabolism. But a small number of oxygen is converted into reactive oxygen species with high oxidative activity, which has a great deal of damage to body cells. And intracellular reactive oxygen species is also confirmed to have a close relationship with the occurrence of many diseases. This article aims to build a genetically encoded FRET-based biosensor for oxidation state of the cell, which can quickly and accurately reflect the oxidation state by fluorescence detection technology.Firstly, we successfully cloned and fused four genes rsrA, sigR, cfp and yfp, and constructed four new recombinant expression plasmids rsrA-cfp-pET-28a(+), sigR-yfp-pET-28a(+), cfp-pET-28a(+) and yfp-pET-28a(+). Then they were transformed into E.coli BL21(DE3).Secondly, four proteins RCP, SYP, CFP and YFP were induced to express by IPTG, then the results were proved to be correct by fluorescence determination. We explored fusion protein expression and purification of RCP and SYP furtherly to improve their quantity and purity.Finally, we studied the functional activity of RCP and SYP proteins,while CFP and YFP proteins was as a reference. We verified the binding activity of RCP and SYP proteins, and their complex response to H2O2. Further more, we got the kinetic curve of RCP and SYP, and calculate the binding constants Kd of1.47×10-6. We also proved the role that Zinc is not required for RCP binding SYP. It was found that in addition to the oxidant H2O2, the reducing agent DTT also had an impact on the combination of RCP and SYP proteins, but contrary to the role of H2O2. The conformation of RCP dealed with H2O2was determined by means of circular dichroism.