Recent studies in traumatic brain injury (TBI) have found a temporal window where therapeutics on the nanometer scale can cross the blood-brain barrier and enter the parenchyma. Developing protein-based therapeutics is attractive for a number of reasons, yet, the production pipeline for high yield and consistent bioactive recombinant proteins remains a major obstacle. Previous studies for recombinant protein production has utilized gram-negative hosts such as Escherichia coli (E. coli) due to its well-established genetics and fast growth for recombinant protein production. However, using gram-negative hosts require lysis that calls for additional optimization and also introduces endotoxins and proteases that contribute to protein degradation. This project directly addressed this issue and evaluated the potential to use a gram-positive host such as Brevibacillus choshinensis (Brevi) which does not require lysis as the proteins are expressed directly into the supernatant. This host was utilized to produce variants of Stock 11 (S11) protein as a proof-of-concept towards this methodology. Variants of S11 were synthesized using different restriction enzymes which will alter the location of protein tags that may affect production or purification. Factors such as incubation time, incubation temperature, and media were optimized for each variant of S11 using a robust design of experiments. All variants of S11 were grown using optimized parameters prior to purification via affinity chromatography. Results showed the efficiency of using Brevi as a potential host for domain antibody production in the Stabenfeldt lab. Future aims will focus on troubleshooting the purification process to optimize the protein production pipeline.

One out of ten women has a difficult time getting or staying pregnant in the United States. Recent studies have identified aging as one of the key factors attributed to a decline in female reproductive health. Existing fertility diagnostic methods do not allow for the non-invasive monitoring of hormone levels across time. In recent years, olfactory sensing has emerged as a promising diagnostic tool for its potential for real-time, non-invasive monitoring. This technology has been proven promising in the areas of oncology, diabetes, and neurological disorders. Little work, however, has addressed the use of olfactory sensing with respect to female fertility. In this work, we perform a study on ten healthy female subjects to determine the volatile signature in biological samples across 28 days, correlating to fertility hormones. Volatile organic compounds (VOCs) present in the air above the biological sample, or headspace, were collected by solid phase microextraction (SPME), using a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) coated fiber. Samples were analyzed, using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). A regression model was used to identify key analytes, corresponding to the fertility hormones estrogen and progesterone. Results indicate shifts in volatile signatures in biological samples across the 28 days, relevant to hormonal changes. Further work includes evaluating metabolic changes in volatile hormone expression as an early indicator of declining fertility, so women may one day be able to monitor their reproductive health in real-time as they age.