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

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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.