Characterization of Anti-TcdB DARPin Disulfide Mutants
Abstract
Clostridioides difficile (C. difficile), the microbe responsible for Clostridioides difficile infection (CDI), is a common nosocomial infection that exerts its pathogenicity primarily by two toxins, TcdA and TcdB. Designed ankyrin repeat proteins (DARPins) are an emerging approach of protein therapeutics to combat disease beyond the limitations of antibiotics and monoclonal antibodies. Disulfide bonds are commonly used in other facets of protein engineering to enhance stability, but their use has not been well documented with DARPins. Previously, the Chen lab engineered DARPins that have been demonstrated to be effective at neutralizing TcdB. However, these DARPins are not protease-stable, which is a barrier for effective delivery in downstream therapeutic contexts. To address this, disulfide bonds were introduced in order to increase the stability of the DARPins and thus increase resistance to protease digestion. The subsequent structure, stability, and neutralization activity are assessed to ascertain the effects of bolstering tertiary structure. The formation of disulfide bonds is confirmed by the comparison of mutants in oxidizing and reducing conditions. Elapsed trials with trypsin and chymotrypsin incubation demonstrate protease stability. Overall, the addition of disulfide bonds is demonstrated to improve stability at minimal cost to neutralization activity. The successful characterization of these disulfide mutants may grant continuing insight into future protein engineering applications and aid the development of a therapeutic anti-TcdB DARPin.
Subject
C. difficileDARPin
disulfide bond
protein engineering
protein therapeutics
protein stability
protease
Citation
Low, Alyssa (2022). Characterization of Anti-TcdB DARPin Disulfide Mutants. Undergraduate Research Scholars Program. Available electronically from https : / /hdl .handle .net /1969 .1 /194361.