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Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.
- Source :
-
Metabolic Engineering . Sep2024, Vol. 85, p116-130. 15p. - Publication Year :
- 2024
-
Abstract
- Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli , in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development. • AutoDC REdox robustly expresses VHHs. • 50% of VHHs expressed exceed the highest reported titer for cytoplasmic expression. • Consistent expression with minimal optimization across various VHH sequences. • Scalable fermentations up to a 1L bioreactor with VHH titers reaching up to 2 g/L. • Efficient autolysis and filtration process simplifies downstream purification. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 10967176
- Volume :
- 85
- Database :
- Academic Search Index
- Journal :
- Metabolic Engineering
- Publication Type :
- Academic Journal
- Accession number :
- 179633700
- Full Text :
- https://doi.org/10.1016/j.ymben.2024.07.012