Department of Bioengineering, Northeastern University, Boston, Massachusetts, USA.
Department of Molecular Biology and Biophysics, UCONN Health, Farmington, Connecticut, USA.
Appl Environ Microbiol. 2020 Apr 1;86(8). doi: 10.1128/AEM.02938-19.
There is a growing need for a highly stable system to allow the production of biologics for diagnoses and therapeutic interventions on demand that could be used in extreme environments. Among the variety of biologics, nanobodies (Nbs) derived from single-chain variable antibody fragments from camelids have attracted great attention in recent years due to their small size and great stability with translational potentials in whole-body imaging and the development of new drugs. Intracellular expression using the bacterium has been the predominant system to produce Nbs, and this requires lengthy steps for releasing intracellular proteins for purification as well as removal of endotoxins. Lyophilized, translationally competent cell extracts have also been explored as offering portability and long shelf life, but such extracts may be difficult to scale up and suffer from batch-to-batch variability. To address these problems, we present a new system to do the following: (i) engineer the spore-forming bacterium to secrete Nbs that can target small molecules or protein antigens on mammalian cells, (ii) immobilize Nbs containing a cellulose-binding domain on a cellulose matrix for long-term storage and small-molecule capturing, (iii) directly use Nb-containing bacterial supernatant fluid to perform protein detection on cell surfaces, and (iv) convert engineered to spores that are resistant to most environmental extremes. In summary, our work may open a new paradigm for using as an extremely stable microbial factory to produce Nbs with applications in extreme environments on demand. It is highly desirable to produce biologics for diagnoses and therapeutic interventions on demand that could be used in a variety of settings. Among the many biologics, Nbs have attracted attention due to their small size, thermal stability, and broad utility in diagnoses, therapies, and fundamental research. Nbs originate from antibodies found in camelids, and >10 companies have invested in Nbs as potential drugs. Here, we present a system using cells of the bacterium as a versatile platform for production of Nbs and then antigen detection via customized affinity columns. Importantly, carrying engineered genes for Nbs can form spores, which survive for years in a desiccated state. However, upon rehydration and exposure to nutrients, spores rapidly transition to growing cells which secrete encoded Nbs, thus allowing their manufacture and purification.
对于一个高度稳定的系统的需求日益增长，以允许按需生产用于诊断和治疗干预的生物制剂，这些生物制剂可以在极端环境中使用。在各种生物制剂中，近年来源自骆驼科单链可变抗体片段的纳米抗体（Nbs）因其体积小、稳定性好而备受关注，具有在全身成像和新药开发方面的转化潜力。使用细菌进行细胞内表达一直是生产 Nbs 的主要系统，这需要经过漫长的步骤来释放用于纯化的细胞内蛋白质，并去除内毒素。冻干的、具有翻译能力的细胞提取物也被探索为提供便携性和长保质期，但这种提取物可能难以扩大规模，并受到批次间变异性的影响。为了解决这些问题，我们提出了一种新的系统来实现以下目标：（i）工程化芽孢形成细菌 ，使其分泌能够靶向哺乳动物细胞中小分子或蛋白质抗原的 Nbs；（ii）将含有纤维素结合结构域的 Nbs 固定在纤维素基质上，用于长期储存和小分子捕获；（iii）直接使用含有 Nb 的细菌上清液在细胞表面上进行蛋白质检测；（iv）将工程化的 转化为对大多数极端环境具有抗性的孢子。总之，我们的工作可能为使用 作为一种极其稳定的微生物工厂来按需生产具有极端环境应用潜力的 Nbs 开辟了一个新的范例。非常希望能够按需生产用于诊断和治疗干预的生物制剂，这些制剂可以在各种环境中使用。在许多生物制剂中，Nbs 因其体积小、热稳定性以及在诊断、治疗和基础研究中的广泛应用而受到关注。Nbs 源自在骆驼科中发现的抗体，超过 10 家公司已经投资于 Nbs 作为潜在药物。在这里，我们提出了一个使用芽孢杆菌细胞作为生产 Nbs 的多功能平台的系统，然后通过定制的亲和柱进行抗原检测。重要的是，携带 Nbs 工程基因的 可以形成孢子，这些孢子在干燥状态下可以存活数年。然而，在重新水合并暴露于营养物质后，孢子迅速转变为生长细胞，分泌编码的 Nbs，从而允许其制造和纯化。
