Have you ever locked yourself in a freezer? Neither have I, but I imagine that it’s something like spending thirty seconds in the lab’s cold room. The microbiologists and chemists of the Lefkowitz lab need the cold room at temperatures near freezing to impede natural processes and preserve the trillions of proteins that line their metallic walls. Dr. Masoudi had to leave for a bit, so I spent the early morning reading up on G-Protein Coupled Receptors and helping Dr. Li Yin, Dr. Masoudi’s co-researcher. Dr. Li Yin was kind enough to let me do a majority of the hands-on work for her protein identification process, but this included heading into the cold room to occasionally refill the plastic column she uses for chromatography with a wash solution (it’s freezing in there). This form of chromatography works by employing an antibody that has a high affinity for the receptor that we want to extract. Everything else in the solution passes through the column without strongly interacting with the antibody in the resin. Eventually, Dr. Li Yin will pass a molecule through the same column that has an even stronger affinity for the antibody than our receptor, removing the receptor from the column. We also spent time grinding the cell membranes of moths – not something I thought I’d write in my lifetime. These moth cell-membranes hold the GPCR that we want, but in order to prepare them for filtration we need to homogenize the cell solutions and eventually mix them with some detergent. These soap molecules will act as a shield for the receptor, sustaining the structures for crystallization.
I also had the opportunity to make some stock solutions and use high-tech pipettes to deliver them into tiny aliquot containers. The LB media that I made is a combination of benzonase, a highly reactive enzyme, and protease inhibitor that prevents the denaturation of helpful cellular proteins. We soon moved on to her Western blots that showed the presence of a receptor using a set of primary and secondary enzymes. The secondary enzyme that was involved actually derives from rabbits! Another one of the enzymes she used was horseradish peroxidase that, yep, comes from horseradish.
A majority of the day was used gathering information to understand these receptors on a broader scale. Dr. Masoudi and I went over the sequence that some of these receptors undergo, from the moment an agonist – adrenaline, morphine, a photon – binds, to the recycling of these receptors in the lysosomes of cells. There’s quite a bit of literature on GPCRs, but barely any information on the mapping of these macro-molecules with X-Ray crystallography, and even less info on our synthetic protein Nb6B9 (FUN FACT: Nb6B9 was originally made from the antibodies of llamas).
Finally, Dr. Masoudi prepared our bacterial colony by taking a set of cells that (hopefully) incorporated the Nb6B9 gene into their DNA. We’ll know for sure that the gene was successfully incorporated after the DNA is sequenced by a lab at Duke, but for now we’re hoping that the colony we’ve set up will grow to produce as much Nb6B9 as possible. To avoid any fungal infections, Dr. Masoudi uses a lamp to ward off aerial imposters, but he also uses ethanol to clean the table. We really only realized how precarious the situation was after we had finished setting everything up.
