Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

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 f

SUBMITTER: Hennigan JN 

PROVIDER: S-EPMC11408108 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature