It is possible to build proteins with desired characteristics and capabilities by fine-tuning protein assembly with an understanding of the dynamics of protein oligomerization. Hetero-telechelic proteins, which are known for their simple and spontaneous reactivity, have been designed and used for a variety of protein assembly structures, however, difficulties in managing protein oligomerization dynamics during production and purification have limited their potential applications. The final structures that result from the processing of the hetero-telechelic proteins after their expression also vary, albeit the relationship between the rate of expression and the protein's structure is still not entirely understood. Herein, we used the SpyCatcher and SpyTag systems, which provide a spontaneous and robust iso-peptide bond capable of withstanding pH and temperature fluctuations, to study the relationship between expression rate and oligomer state. To see the various protein assembly structures, we also changed the flexibility of the protein segment that was placed between SpyCatcher and SpyTag. We wanted to create broad guidelines for this work. We enhance biomimicry and the creation of custom protein-based materials with particular functionality by revealing the complex link between protein expression and structure.

Per- and poly-alkyl-substances (PFAS) are synthetic chemicals that are introduced to the environment by manufacturing companies. These chemicals persist in the environment for extremely long periods of time. Known as “forever chemicals,” PFAS exposure renders the exposed to test positive for PFAS for their lifetime. Recent research suggests exposure to PFAS results in long term health effects, such as immune system suppression and lowered response to vaccinations. Since widespread usage of PFAS has been increasing yearly and with studies showing the adverse impact on health, a need for regulatory guidelines is now of major importance. In this study, we look at how the varying types of PFAS effects mast cell degranulation rates. Concentration and length of treatment are also analyzed in its affects on the results. PFOA, PFAS, PFHS, PFBS and GENX, were used as treatment groups within this experiment. Rosa Mast cells were exposed to concentrations of the different types of PFAS at 1.0, 10. and 25. μM for 24 and 48 hr. time periods. The treated cells were then “activated” by exposure to a positive control of silver nanoparticles, known to cause activation by the MRGPRX2 receptor. β-Hexosaminidase levels were recorded and analyzed against the treatment group treated strictly with silver nanoparticles. Additionally, the results were compared to treatment groups at varying concentrations to identify if concentration had an impact on results. Furthermore, the results for the 24 and 48 hour time thresholds were compared against each other. The results suggest PFAS has an overall effect on mast cell degranulation. The results vary for each type of PFAS treatment. Certain PFAS, such as PFOA is seen to amplify activation. While certain PFAS, such as GENX is seen to repress activation. Overall, varying the concentrations of PFAS didn’t yield statistically different effects within the treatment groups. However, the time threshold of how long cells were treated for did impact the results obtained. Exposure at 24 hours yielded results that weren’t statistically different then the 4880 control. This suggesting that longer periods of exposure cause a greater effect on mast cell activation. In conclusion, PFAS exposure to mast cells has an impact on the rate of betahx release. PFAS was seen to either increase mast cell degranulation or suppress it, depending on the specific PFAS. While concentration wasn’t seen to impact results, time period did. The future direction of this study is to identify if PFAS has an effect on the glycolysis of the treated mast cell. As well as identifying if cytokine release occurs after mast cells undergo PFAS exposure.

The main purpose of this project is to thoroughly express a recombinant fusion protein known as GFP-SUMO1, as a cleavage control for the eventual activity testing of a nuclease known as Cas13a. Currently in the field, Cas13a is used for diagnostic rapid testing by closely recognizing and matching nucleic acids in a given sample. Since GFP-SUMO1 is a SUMO fusion protein, SUMO-specific proteins such as ULP1, can eliminate this SUMO tag. This tag is known as a fusion tag that is present in the protein, which partly acts as an affinity tag by making the target protein easier to detect and purify. The overall goal is to determine if a recombinant protein called ULP1 can efficiently cleave off inessential tags in GFP-SUMO1. GFP-SUMO1’s expression and purification will give more insight on ULP’s functions and interactions with SUMO-specific proteins, which will be beneficial for Cas13a’s purification. The first step before following through GFP-SUMO1’s expression is isolating its high-purity DNA from the bacterial strain it was initially stored in through a miniprep procedure. Once isolated, the plasmid was further investigated in order to find its distinct accommodations through plasmid sequencing. From analyzing the sequence, the plasmid was found to have a T7-based expression vector, which relies on the specificity of a particular inducing agent, in this case IPTG, and strain of BL21(DE3) pLysS (BDP) competent cells needed for expression. By expressing this protein, site-specific cleavage from the SUMO tag is possible, while also having the protein at hand for Ni-NTA purification.