By adding the protein that binds to the Ni21-NTA to the affinity column, we were able to collect the liquid that went through. We ran the SDS-Page Gel with thirteen chosen samples to verify the results of the protein purification. It turns out, we successfully purified pET28-mlrA1, indiciating that it can be devoted to degrading MC-LR.
As you can see from the picture, we use N-Dodecyl-β-D-maltoside, which is also known as DDM, to transfer the target membrane protein mlrA from the supernatant to the membrane of DDM. Since DDM provides the membrane which mlrA depends on, with the DDM concentration increases, the concentration of the mlrA in the supernatant decreases to zero. That is the last step of protein purification.
After centrifuge the solution, to further purify the protein, we put Ni21-NTA with the supernatant and incubate overnight. The mlrA will be combined with the specific binding sites on Ni21-NTA. Then, using the method of gradient elution, while the amount of imidazole, which is a competitive agent in this step, increases, it will compete with mlrA on the binding sites of the Ni column. Therefore, the mlrA is gradually elated.
Enzyme Function Test
High Performance Liquid Chromatography (HPLC)
To quantify whether MC-LR can be degraded from the samples, we conducted a control variable experiments, and the following are the data we got:
The original HPLC peak map of MC-LR at the concentration of 0.5mg/ml
The HPLC peak map we get when the concentration between MlrA and MC-LR is 9:1
1. By adding the protein that we have designed, and letting it react with the MC-LR, we find that they successfully reacted just as we expected.
2. From the HPLC results, we find that as the ratio of the mlrA we have designed increases, the percentage of the reaction product increases and the amount of MC-LR decreases consequencely. This result successful indicates that the protein mlrA we have designed is able to react with MC-LR and release the product which is 160 times less toxic than MC-LR, as we mentioned in the Background.