Affinity maturation via in vitro evolution at the lab bench-top

我们建议开发一种完全自动化的台式系统，可以生成紧密结合的蛋白质支架，称为纳米体，针对任何目标酶。我们方法的新颖之处在于能够使用完全体外的方法快速“进化”这些纳米体。我们设想，用户将纯化的蛋白质注射到这个台式系统中，紧密结合的Nbs将在几天内迅速“进化”。我们的总体策略是模仿自然界中发现的遗传优化主题，并在创新的无动物系统中生产高度特异性的纳米体探针。该方法将包括结合一些较新的、更危险的技术，如mRNA展示和基于PCR的DNA重组方法，以及利用更成熟的技术，如无细胞蛋白表达和PCR扩增。该系统的产物将是针对目标酶和相应DNA的紧密结合的多克隆纳米体，可用于产生更大量的单克隆纳米体。Nbs可以直接用于日常实验室应用，目前由传统的多克隆抗体完成。我们设想，纳米体在实验台上的快速发现、优化和生产将产生与聚合酶链反应（PCR）机器几十年前对分子生物学产生的革命性影响相同的影响。这种创新的方法将使广泛的生物医学研究界能够快速生成高度特异性的生物探针、药物和其他用于基础科学研究的分子工具，同时避免了目前使用动物产生抗体所带来的经济和时间成本。我们建议开发一个完全在体外的台式系统来从头创建纳米体，使科学界能够在自己的实验室中快速生成药物、生物探针和分子工具。这个提议的想法不需要使用动物，并且可以比目前的体内方法至少快一个数量级。

We propose to develop a fully automated bench-top system that can generate tight-binding protein scaffolds called nanobodies against any target enzyme. The novelty of our approach lies in the ability to rapidly "evolve" these nanobodies using completely in vitro methods. We envision that the user injects a purified protein into this bench-top system and tight-binding Nbs will rapidly "evolve" within days. Our overall strategy is to mimic the genetic optimization themes found in nature and produce highly specific nanobody probes in an innovative animal-free system. The approach will include combining some newer, more risky technologies such as mRNA display and PCR-based DNA recombination methods as well as utilizing more established techniques like cell free protein expression and PCR amplification. The products of the system will be tight-binding polyclonal nanobodies against the target enzyme and the corresponding DNA, which can be used to generate larger quantities of monoclonal nanobodies. The Nbs can be directly used in everyday laboratory applications currently accomplished with traditional polyclonal antibodies. We envision that the rapid discovery, optimization, and production of nanobodies at the bench will have the same revolutionary impact that polymerase chain reaction (PCR) machines had on molecular biology decades ago. This innovative method will allow the broad biomedical research community to quickly generate highly specific biological probes, drugs, and other molecular tools for basic scientific research while circumventing the economic and time costs currently associated with the use of animals to generate antibodies. We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods. PUBLIC HEALTH RELEVANCE: We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods.