Part 2 -Experiment Exploring Enzyme Activity: Research at Institute of Cancer Research at New York

In this section, we get into the actual experiment and its details. I learned a lot of new things from that week and I hope by the end of this experiment, you will learn a lot more new things too!

1) Bacterial Transformation

We started by adding the plasmid to the E. coli bacteria. We had a negative control, a positive control, and the mutated S8D plasmid plates. Then, we added in the Kanamycin antibiotic as a resistance. After that, we moved the E. coli bacteria from the plates to the media. It was left to grow in the flask with the kanamycin addition for 3 hours in total.

Over time, the flask was measured until 0.4-0.6 density was reached. After optimal density was reached, the cells were treated with IPTG which allows for RNA polymerase to express genes. The pictures below show the measurements over time.

2) Protein Purification

After inducing gene expression, the next crucial step was to extract the target protein from the E. coli cells. Once the cells were treated with IPTG, we spun them down using centrifugation to pellet the cells. From here, we moved on to extracting the protein.

To purify malate dehydrogenase 2 (MDH2), we prepared three key buffers:

• His-binding buffer: This buffer ensures that proteins containing histidine tags (like MDH2 in our experiment) bind tightly to the affinity column.

  
  

• Wash buffer: Used to wash away any non-specific or contaminant proteins, leaving behind only the protein of interest.

  
  

• Elution buffer: Finally, the elution buffer was applied to release MDH2 from the column and collect the purified enzyme.

  
  

We used these buffers during affinity chromatography, allowing us to isolate MDH2 while removing unwanted proteins. This step was critical to ensuring we worked with a highly purified form of MDH2 for further analysis. The picture below is the tool we used to separate the MDH2.

3) Bradford Assay

After successfully purifying MDH2, our next step was to measure how much of the protein we had. To do that, we performed a Bradford Assay, which is a colorimetric test that tells us the concentration of protein based on its binding to Coomassie Brilliant Blue dye. We started by breaking open the E. coli cells using a centrifuge, which allowed us to extract the proteins. Then, we used the Bradford Assay to estimate the concentration of MDH2 in our sample.

To get accurate results, we created a standard curve using Bovine Serum Albumin (BSA). We used the absorbance readings from our samples and compared them to the BSA standard to calculate the MDH2 concentration. We generated a graph that shows our findings. Below, you can see the final Bradford Assay graph we created based on the multiple readings we took.

Bradford Assay final graph based on multiple readings

4) Protein Gel

After purifying the MDH2 proteins through affinity chromatography, the next step was to confirm the presence and purity of the extracted proteins using an SDS-PAGE gel. This technique allows us to separate proteins based on their molecular weight, ensuring that we’ve successfully isolated MDH2 from the other proteins in the E. coli cells. The picture below is the SDS-PAGE gel process.

Results and Conclusion:

This is a graph of how the NADH concentration decreases over time as the enzyme and substrates react. This data provides crucial insight into MDH2’s enzymatic role and its efficiency in catalyzing reactions.

These gel results show the MDH2 band being the most intense, especially in E2 and E3. This confirmed that these fractions contained a high concentration of purified hMDH2 S8D.

We originally thought that the mutant form of MDH2 (S8D), which mimics phosphorylation, would perform better or faster than the normal (wild-type) MDH2 enzyme. However, our experiment showed the opposite. The wild-type enzyme worked much faster. The mutant was 8.75 umol/min/mg while the wild-type was 200 umol/min/mg. Since the specific activity of the mutant was lower than that of the wild type, this falsifies our hypothesis. This means the mutant to longer to convert NADH to NAD+ compared to the wild type.

In conclusion, our hypothesis (phosphorylation of S8 makes the interaction of NADH stronger and increases the rate of the forward reaction) was refuted. The wild-type reaction rate is significantly higher than the mutated reaction rate with a difference of 191.25 umol/min/mg.

We believe that this was caused by structural differences outside of the mutation itself.

The shape of the active site may have been altered, causing the reaction rate of the mutated MDH2 to be slower than predicted. We could confirm this through X-ray crystallography. This technique would let us see if the active site or other parts of the enzyme were altered by the mutation, which could explain why the reaction rate of the mutant MDH2 was so much lower than the wild type.

Even though the hypothesis was refuted, it gave a lot of insight into how to approach this experiment next time. I learned a lot by doing this lab and it gave me first-hand experience on how to use all the equipment and how each process works together as a whole. I found this experiment to be very fun and I had a good team to work with. The professor was also very helpful in explaining everything to us and showing us how to do the lab. She offered lots of guidance. I will definitely try to do more experiments like this one in the near future!