To start off my project at the SGC, my supervisor, Dr. Rachel Harding, and I produced and purified three proteins. These proteins were RAB1A, RAB1B, and MECP2. Both RAB1A and RAB1B have functions in the initiation of autophagy, which is a cellular system that helps clean out old debris and malfunctioning parts. MECP2 is a protein that can interact with DNA, changing the levels of expression of several genes. The purifications of RAB1A and RAB1B were successful, although MECP2 could not be adequately purified. These three proteins were hits with the BioID experiment I mentioned in my previous post.
To begin, we took E. coli cells and transformed them with plasmid DNA. The transformation process introduces the bacteria to the genetic instructions on how to make the protein, the cells were left to grow for about 24 hours. By the end, we had 6 L of liquid culture for both RAB1A and RAB1B as well as 4 L of MECP2. From this liquid culture, we collected the cells and proceeded to break them open using sound energy via a process called sonication. After opening the cells, we were able to purify our protein using two methods. The first was affinity chromatography, which allows the binding of a specific tag we put on our protein. To further refine the protein sample, we used size-exclusion chromatography, which is a process that separates proteins based on their size. For a more in-depth explanation of the methods used to purify RAB1A, RAB1B, and MECP2, follow the links to the Zenodo posts about each protein.