Team:TAS Taipei/Demonstrate
Demonstrate
To confirm that the protein denaturation in our cataracts fish lens model also happens to cataractous human lenses, we designed and cloned a CRYAB construct to test the protein denaturation of human lens proteins. We ran a protein gel with the lysate of CRYAB with and without H2O2 and found that the more H2O2 added, the lighter the original CRYAB bands are and darker the higher bands become. This demonstrates that CRYAB is aggregated and clumped due to H2O2 when cataracts is formed in human lenses. Furthermore, we showed that our delivery prototype can be used to prevent and treat cataracts in our fish lens model by adding GSH and 25HC containing nanoparticles into our fish lens solution.
Demonstrating Function using Human Proteins
CRYAB is one of the main proteins in the human lens that aggregates to form cataracts. In our fish lens model, we observed protein aggregation after adding H2O2. However, we also wanted to make sure that the fish model results represent what happens with human crystallin proteins, so we designed a CRYAB construct (figure 2.12).
The final CRYAB construct contains similar components, except the first part of the open reading frame is replaced by CRYAB. CRYAB cDNA was ordered from Origene. We designed primers that were synthesized by Tri-I biotech to clone CRYAB into a Biobrick backbone, similar to how the GSR construct was made. Sequencing results (Tri-I Biotech) show that the construct was correct, and we confirmed protein expression of both CRYAB (without a poly-his tag; yellow asterisk in (figure 2.13) and CRYAB-HIS (blue asterisk in figure 2.13).
To test that CRYAB aggregates in response to H2O2, we cultured bacteria expressing CRYAB and CRYAB-HIS. Liquid cultures were grown overnight and detergent was added to lyse the bacteria cultures. Different concentrations of H2O2 were added to the lysates and a protein gel was prepared (figure 2.13). With increasing concentration of H2O2, the original band for CRYAB-HIS (blue asterisk) becomes lighter as higher bands (indicated by the blue bracket) increase in concentration. This shows that H2O2 has the same effect on human crystallin proteins, causing them to aggregate and clump.
Demonstration of Our System
In a final experiment, we wanted to test if our delivery prototype can actually be used to prevent and treat cataracts in our model. As shown in figure 3.12, we first solubilized fish lens proteins and treated these samples with H2O2 to simulate cataracts. Nanoparticles encapsulating the compounds GSH or 25HC were made. Next, we added the nanoparticles into our cataracts model to test for prevention and treatment effects. We compared the absorbance values after 48 hours.
After adding H2O2 to the lens solution, absorbance values increased. However, when GSH-containing nanoparticles were also added, the absorbance values remained unchanged (figure 3.13). Similarly, when 25HC-containing nanoparticles were added, the increase in absorbance was significantly lower than with only H2O2 (figure 3.14). These result show that, in our cataracts model, the compounds carried inside nanoparticles were released and effective at treating and preventing cataracts.
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