Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

Abstract

AbstractNanobodies 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 inE. colican 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 ofE. 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, in combination with dynamic expression of chaperones and lysis enzymes needed for purification. 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.