Part:BBa_K1456003

Superoxide dismutase-1 (SOD-1)

• SOD is a powerful and essential antioxidant enzyme which converts and scavenges free radicals and reactive oxygen species (ROS) into hydrogen peroxide (H2O2), slightly less harmful compound in order to convey it to the second step reaction which detoxifies H2O2 into water. (Batelli et al. 1972; Baudry et al. 1993; Gonzalez et al. 1995; Doctrow et al. 1996)
• In our body cells, there are three different subtypes of SOD enzyme in different cellular levels. SOD-1 exists in cytoplasm, SOD-2 in mitochondria and SOD-3 is in extracellular matrix. We have chosen SOD-1 to take advantage of easy interference and activity capacity due to its location. Additionally, there is evidence in the literature for the synthetic production of SOD-1 enzyme by transfecting human cells. (Shuvaev et al. 2013)

Transfected Tet off - pTRE and Tet off - pTRE-SOD1 vectors were subjected to SOD assay and measured using the spectrophotometer at 560 nm. Cells which have the Tet off – pTRE SOD1 gene present can degrade the oxygen radicals 21 times faster. SOD assay was performed for the proteins obtained from the transfected SOD1 visible colour change could be seen.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 327
  

hSOD-linker-hCatalase

Description

Considering the fact that not all UV radiation could be absorbed by MAAs and could still result in increased oxidative stress, we decided to break down ROS (Reactive Oxygen Species). Therefore, we decided to produce human Superoxide Dismutase 1 (hSOD) and human catalase (hCatalase). hSOD possesses the ability to convert superoxide (O2-), the main form of ROS, into hydrogen peroxide. However, hydrogen peroxide is also harmful to our body, and catalase provides a perfect solution to this problem as it could break down hydrogen peroxide into water.

Usage and Biology

We used the old brick hSOD (BBa_K1456003) as the basis for fusing the hCatalase gene to get the SHC fusion protein by connected these two genes with a flexible linker (Figure 1). Use promoter pT7 and terminator pET28a to express hSOD-linker-hCatalase (SHC) and transformed the plasmids into E. coli BL21 Strain.

Figure 1: The design of fusion protein SHC

Characterization

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 (Figure 2). The result shows the production of hSOD, hCatalase and SHC are achieved in concentrations of 270mg/L, 240mg/L and 400mg/L respectively.

Figure 2: Production, purification and recovery of proteins

Under the same conditions, we set up assessments for hSOD, hCatalase and fusion protein hSOD-linker-hCAT. Quantitatively, our results showed that separate hSOD and hCatalase had activities of 3.00U/mg and 128.85U/g, respectively. In comparison, hSOD and hCAT in our fusion protein SHC produced activity levels of 8.04U/mg and 313.04U/g (Figure 3 & 4). By calculation, fusion protein SHC enhanced SOD activity by 168% and catalase activity by 143% compared with single protein.

Figure 3：SOD assay kit with WST-8 test enzyme activity of hSOD, hCatalase and SHC. Effects of hSOD, catalase, and SHC fusion protein at removing ROS(A). The concentration of yellow dye WST-8 formazan can indicate the enzyme activity of SOD, the lighter the color, the higher the enzyme activity(B). Absorbance value detection of WST-8 formazan (C).

Figure 4: Catalase assay kit test of hSOD, hCatalase and SHC. Effects of hSOD, hCatalase, and SHC fusion protein in removing H2O2(A). The concentration of Red color can indicate the enzyme activity of catalase, the lighter the color, the higher the enzyme activity(B). The standard curve of hydrogen peroxide(C). The remaining levels of hydrogen peroxide (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(Figure 5). By calculation, fusion protein SHC enhanced SOD activity by 168% and catalase activity by 143% compared with single protein.

Figure 5: 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 hSOD group has little amount of H2O2 present, while the hCatalase and the SHC groups finished the H2O2 degradation completely (C).

Overall, this confirmed our engineering hypothesis as the efficacy of the fusion protein is higher than the two enzymes working separately.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]