Rapid purification of recombinant proteins by protein nanoparticle crosslinking and light-responsive nanobodies

对以蛋白质为基础的新药物的需求不断增加，加速了对大规模生产更有效工艺的需求。获得具有成本效益和高效的蛋白质纯化过程是最紧迫的挑战之一。该项目将通过开发一种基于可逆的、光响应的蛋白质捕获的创新策略来解决蛋白质纯化方面的长期挑战。蓝光和红光可以用来控制蛋白质的结合、沉淀和所需纳米支架分子的回收。与传统的层析方法以及其他基于亲和力的方法相比，该策略具有许多操作上的优势。光响应纯化平台还可以被定制为捕获几乎任何感兴趣的天然蛋白质。该项目的成果有可能改变工业蛋白质纯化过程，因为它消除了对层析的需要，而层析成本往往超过总蛋白质生产成本的50%。效率的提高预计将大幅降低治疗性蛋白质的成本，并对国家的健康和福祉产生积极影响。高中生参与暑期研究的机会，加上本科生延长的研究机会，将提供宝贵的培训机会，并有助于发展未来的STEM工作力量。该计划的总体目标是开发一个新的亲和沉淀平台，通过将最先进的光遗传工具与纳米支架亲和沉淀结合在一起，形成一个新的变革性框架，用于天然蛋白质的纯化。有条件的纳米体将被蓝光光遗传激活，以产生可切换的靶蛋白结合特性。一个红光响应域将被插入光体修饰的纳米支架中，以使其能够恢复。该项目的目标包括(I)设计用于蓝光可切换抗原结合的最佳光体，(Ii)用不同的捕获光体基元装饰纳米笼以捕获和释放目标蛋白，(Iii)共修饰纳米笼以用于红光激活的聚集，以及(Iv)评估目标蛋白的纯化和纳米笼再生的效率。蛋白质纯化平台操作简单，因为蛋白质捕获/释放和纳米支架回收可以使用两个正交的蓝光和红光触发器独立控制。纳米体基序和光响应蛋白质元件都是高度模块化的，使这种纯化策略能够用于靶向许多感兴趣的天然蛋白质。由于该平台易于操作、与小型化兼容、消除了层析，以及灵活性和在很大程度上与目标无关的过程，该平台可以改编为高通量格式。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The increasing demand for new protein-based drugs has accelerated the need for more effective processes for their large-scale production. Access to cost-effective and highly efficient protein purification processes is among the most pressing challenges. This project will address the persistent challenges in protein purification by developing an innovative strategy based on reversible, light-responsive protein capture. Blue and red light can be used to control protein binding, precipitation, and recovery of the required nanoscaffold molecules. This strategy has many operational advantages over purification by conventional chromatographic methods as well as other affinity-based methods. The light-responsive purification platform can also be tailored to capture virtually any native protein of interest. The outcomes of this project have the potential to transform industrial protein purification processes by eliminating the need for chromatography, the cost of which often exceeds 50% of the overall protein production costs. The efficiency gains are expected to substantially reduce the cost of therapeutic proteins and positively impact the health and wellbeing of the nation. Engagement of high school students in summer research opportunities, combined with extended research opportunities for undergraduate students, will provide valuable training opportunities and help develop the future STEM workforce.The overall goal of the proposal is to develop a new affinity precipitation platform for recombinant protein purification by combining state-of-the-art optogenetic tools with nanoscaffold-based affinity precipitation into a new transformative framework for native protein purification. Conditional nanobodies will be optogenetically activated by blue light to create switchable target protein binding properties. A red light-responsive domain will be inserted in the optobody-modified nanoscaffold to enable its recovery. The project objectives include (i) design optimal optobodies for blue-light switchable antigen binding, (ii) decorate the nanocages with different capturing optobody motifs for target protein capture and release, (iii) co-decorate the nanocages for red light-activated aggregation, and (iv) evaluate the target protein purification and nanocage regeneration efficiency. The protein purification platform is operationally simple since protein capture/release and nanoscaffold recovery can be independently controlled using two orthogonal blue and red light triggers. Both the nanobody motif and the light-responsive protein elements are highly modular, enabling this purification strategy to be used to target many native proteins of interest. This platform can be adapted into a high-throughput format owing to the ease of operation, compatibility for miniaturization, elimination of chromatography, and the flexibility and largely target-independent process.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.