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Eukaryon

The Road to Crystallizing the hERG Pore

Lindsay Hartup
Department of Biology 
Lake Forest College 
Lake Forest, IL 60045 

“The O.D. is dropping again,” I told Dylan after what felt like the fiftieth time we tried to ferment the hERG pore in C43 E. coli cells this sum- mer. “Well shoot,” he said. Regardless of what we seemed to do, we could not get this potassium channel to overexpress. We tried transforming into different batches of lab made-competent C43 cells, using different plates to inoculate the starter culture, and even pumping additional antibiotics during the fermentation, but nothing worked. It was not until the day that we decid- ed to try using fresh DNA that our results changed. It was a simple fix, but it was something that took us weeks to figure out. Once I made up a new hERG pore DNA stock, transformed the DNA into E. coli, and inoculated a starter culture using this plate, we had a successful fermentation run and were able to harvest 60 grams of cells.

This summer I had the opportunity to work in the laboratory of Dr. Adrian Gross at Rosalind Franklin University. Dr. Gross’ research centers on potassium channels, a family of membrane proteins vital to maintain- ing resting membrane potentials and mediating action potentials in excit- able cells. One potassium channel of particular importance is encoded by hERG, the human ether-a-go-go related gene. This voltage-gated channel is known for maintaining normal electrical activity in the heart by mediating the repolarizing IKr current in the cardiac action potential (Sanguinetti et al., 1995).

Mutations in hERG or blocking by drugs can induce long QT syndrome, a condition that predisposes individuals to developing fatal ar- rhythmias, such as torsade de pointes (TdP) and sudden cardiac death (Sanguinetti & Tristani-Firouzi, 2006). TdP is characterized by a length- ened QT interval on an electrocardiogram, and while it may revert to a normal sinus rhythm, it can also degenerate into fatal ventricular fibrillation due to rapid increases in ventricular heart rate. Despite the importance of the hERG channel in medicine and drug design, the channel has never been crystallized in its entirety. This in part is due to the protein’s instability, which is caused by the exterior voltage sensors being highly mobile and flexible. However, the pore of the channel is much more stable. As such, studies that can be done with the pore’s crystal structure would be much more accurate and reliable than those done with the entire channel. In fact, structural studies of the pore may reveal information about binding and protein conformational changes that are currently not understood. In turn, this can assist pharmaceutical companies in designing drugs and physi- cians in understanding cardiac arrhythmias. Given the protein’s amino acid sequence, I hypothesized that DNA synthesized de novo could be used to overexpress, purify, and ultimately crystallize the hERG pore.

After finally getting a successful fermentation run this summer, I began protein purification. Potassium channels are inherently difficult to purify because they are membrane-bound proteins. Because these chan- nels easily aggregate, it is difficult to solubilize them. It was notoriously difficult to keep this particular protein in solution due to its instability at room temperature. Thus, unlike with other proteins, this purification had to be done entirely at four degrees Celsius. Additionally, after much trial and error, I also found that it was necessary to do a batch elution in lieu of the column elution to prevent the protein from precipitating. Doing so was crucial because once the protein precipitated, the capability for future structural studies was depleted. Once I figured out the importance of these conditions in the purification process, I was able to successfully purify the protein.

To verify the identity of the purified protein, gel filtration, SDS gel electrophoresis, mass spectrometry, and N-terminal sequencing were used. While the gel filtration yielded results that could be expected if we had purified the hERG pore, N-terminal sequencing revealed that this was not the case. Rather, two proteins were purified, of which neither had a polyhistidine tag on the N-terminus. Thus, while the synthesized DNA could be stably transformed into E. coli, the hERG pore could not be overex- pressed.

Initially I was discouraged by these results. I felt like I wasted everyone’s time because the project I spent entire summer working on did not work. However, after some time of reflection I realized this was not the case. Despite the channel not being able to be overexpressed in C43 cells using this DNA construct, there are still many options moving forward with

the hERG pore. Future studies can be done which implement this DNA construct in other model organisms, such as yeast or insects. Because this is a mammalian channel, it is possible that it is unable to be grown in E. coli. Additionally, the codons could be further optimized for E. coli, and the construct can be implemented into other strains of E. coli. While the construct did not work in C43, C41 or SG1 cells, it is possible it may work in other strains such as Origami, DE52, or Orai. Because the benefits from overexpressing, purifying, and crystallizing the hERG pore are so great, efforts should continue to be made to do so.

 

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