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.

中文翻译：

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通过 2 阶段动态控制实现纳米抗体的可扩展、稳健、高通量表达和纯化


纳米抗体是单域抗体片段，已被广泛用作诊断和治疗剂以及研究工具。然而，与许多蛋白质一样，获得纯 VHH 可能很费力且不一致。由于保守的二硫键减少导致不适当的折叠和不溶性聚集，大肠杆菌中的高水平细胞质表达可能具有挑战性。我们报道了一种系统工程方法，该方法利用大肠杆菌的工程菌株，结合两阶段工艺和简化的下游纯化，实现改进的、稳健的、可溶性细胞质纳米抗体表达，以及快速的细胞自溶和纯化。这种方法依赖于对细胞质还原电位的动态控制，结合裂解酶进行纯化，还可以整合蛋白质折叠催化剂的动态表达。总的来说，该工程系统可实现更稳健的生长和蛋白质表达，从而实现高效、可扩展的纳米抗体生产，以及从高通量微量滴定板到常规摇瓶培养和更大型仪器化生物反应器的纯化。我们预计该系统将加快 VHH 的发展。