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The sunburn repair part of this year's LINKS project required the removal of oxidative free radicals. We used the old brick hSOD (BBa_K1456003) as the basis for fusing the hCatalase gene to get the SHC fusion protein (BBa_K4273000). Throughout the experiment, we briefly mention the functions of two enzymes, hSOD and hCatalase. We connected these two genes by a flexible linker. We expressed the separate enzyme and fusion protein with the E.coli BL21 strain and found that they both have high water solubility and are easy to extract the active protein. The result shows the production of hSOD1, hCatalase and SHC are achieved in concentrations of 270mg/L, 240mg/L and 400mg/L respectively.
Figure 1: Design and expression of hSOD, hCatalase, and fusion protein hSOD-flexible linker-hCatalase (SHC). We use promoter pT7 and terminator pET28a to express hSOD, hCatalase and SHC and transformed these plasmids into E. coli (A). After we synthesize SOD and catalase vectors, we used PCR to amplify the plasmid fragments (B). We created SHC using Gibson assembly, and through colony PCR and sequencing, determined that construction was successful (C). We induced the expression of SHC, hSOD, and hCatalase and obtained purified protein samples.
Our team tested the function by ELISA. We used the kit for hSOD and the enzyme activity reached 3.00U/mg. For hCatalase, the enzyme activity reached 128.84U/g. Under the same conditions, our fusion protein reached 8.05U/mg over hSOD enzyme activity and reached 313.04U/g over hCatalase enzyme activity, which proved that the fusion protein has better performance.
Figure 2：Effects of hSOD, catalase, and SHC fusion protein at removing ROS. In order to test the enzyme activity of hSOD, catalase and SHC in converting superoxide (O2-), we tested WST-8 formazan concentrations, which would be decreased if hSOD is present (A). The WST-8 operating fluid was tested after incubation for 30 minutes, and the color was recorded (B). A lighter color represents lower WST-8 formazan concentration. The value of WST-8 formazan was found and summarized into a bar graph, with SHC being lowest, indicating it was most efficient in removing ROS.
Figure 3： Effects of hSOD, hCatalase, and SHC fusion protein in removing H2O2. In order to test the enzyme activity of hSOD, catalase and SHC in converting hydrogen peroxide into water and hydrogen, we tested H2O2 concentrations. If catalase activity, peroxidase activity decreases, thus showing a less red color. The samples were tested after incubation for 30 minutes, and the color was recorded (B). A lighter color represents higher enzyme activity. The standard curve of H2O2 concentration to absorption was found (C). Using the standard curve, we calculated the remaining levels of H2O2 in the sample after catalase and SHC were added (D). SHC was shown to have greater enzyme activity.
Furthermore, Xantine Oxidase can catalyze the production of ROS by Xanthine, and by adding our fusion protein SHC into this system and reacting for 60 minutes, we discovered that the ROS and H2O2 produced by the reaction are all degraded. Therefore, the results indicate that improvements are made in our experiment.
Figure 4： ROS produced via the oxidation of Xanthine breakdown by hSOD1, hCatalase and SHC. SOD converts the ROS into Hydrogen Peroxide, whereby hCatalase then breaks it down completely. SHC, possessing functions of both enzymes, is able to complete the whole chain of reaction (A). After 60 minutes of reaction, and the color was recorded (B). The hSOD1 group has little amount of H2O2 present, while the hCatalase and the SHC groups finished the H2O2 degradation completely (C).