Matching Items (2)

Filtering by

Clear all filters

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

Description

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.

Contributors

Agent

Created

Date Created
2019-05

161638-Thumbnail Image.png

Control of Tissue Homeostasis, Tumorigenesis, and Degeneration by Coupled Bidirectional Bistable Switches

Description

The Hippo-YAP/TAZ signaling pathway plays a critical role in tissue homeostasis, tumorigenesis, and degeneration disorders. The regulation of YAP/TAZ levels is controlled by a complex regulatory network, where several feedback loops have been identified. However, it remains elusive how these

The Hippo-YAP/TAZ signaling pathway plays a critical role in tissue homeostasis, tumorigenesis, and degeneration disorders. The regulation of YAP/TAZ levels is controlled by a complex regulatory network, where several feedback loops have been identified. However, it remains elusive how these feedback loops contain the YAP/TAZ levels and maintain the system in a healthy physiological state or trap the system into pathological conditions. Here, a mathematical model was developed to represent the YAP/TAZ regulatory network. Through theoretical analyses, three distinct states that designate the three physiological and pathological outcomes were found. The transition from the physiological state to the two pathological states is mechanistically controlled by coupled bidirectional bistable switches, which are robust to parametric variation and stochastic fluctuations at the molecular level. This work provides a mechanistic understanding of the regulation and dysregulation of YAP/TAZ levels in tissue state transitions.

Contributors

Agent

Created

Date Created
2021