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Optimizing Recombinant Protein Production for Domain Antibodies: Proof-of-Concept

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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

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.

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2019-05

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Characterizing the Mucin Layer in Eosinophilic Esophagitis

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The purpose of this project was to characterize the mucin layer in patients with eosinophilic esophagitis (EoE). EoE is a chronic disease that is characterized by eosinophilic inflammation in the esophagus. The current diagnosis and standard of care for patients

The purpose of this project was to characterize the mucin layer in patients with eosinophilic esophagitis (EoE). EoE is a chronic disease that is characterized by eosinophilic inflammation in the esophagus. The current diagnosis and standard of care for patients with EoE is less than ideal. Diagnosis is highly invasive as it requires histological confirmation of eosinophilic inflammation in the esophagus, the patient must undergo an upper endoscopy to obtain the tissue sample. The histology as determined by the pathologist is subjective not quantitative which causes significant error in diagnosis. The current treatment methods are dietary therapy or corticosteroids, which require significant cost and time. The pathology of EoE is largely unknown, though it is known to involve allergic inflammatory and type-2 cytokine-mediated responses. Past studies have determined the genetic expression of mucins to be varied in the esophagi of EoE patients using RNA sequencing techniques. The varied expression of mucins in the esophagi of EoE patients has not been validated at the protein level. This study sought to better define mucin protein expression, specifically that of MUC1, MUC4, and MUC7, in the esophagi of EoE patients (n=4) and control patients (n=3). This was accomplished using histological staining. The tissue samples were stained for eosinophil peroxidase (EPX) in order to visualize the eosinophils, which are a pathological marker of EoE. The results of this study showed a qualitative increase in the protein expression of MUC4 in patients with EoE, indicating that MUC4 may play a protective role in the body’s defense against EoE. MUC1 and MUC7 staining showed no pattern. This study defined the conditions necessary for precise staining of esophageal tissues with the MUC4 8G7 antibody. The orientation of the tissue samples on the slides and the small sample size created significant difficulty in analysis and inhibited quantitative analysis. Future studies with tissue orientation standardization and greater sample size are needed to confirm the findings of this study. If verified, the increase of MUC4 protein expression in patients with EoE has implications for EoE diagnostics and therapeutics.

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Date Created
2020-05