SBIR Phase I: Synthetic biology platform for production of stabilized high-value proteins

这个小企业创新研究（SBIR）第一阶段项目的更广泛的影响/商业潜力是开发使用工程细菌的技术，以提高两个大市场的蛋白质稳定性：治疗性蛋白质和工业酶。用作治疗剂的蛋白质在人血浆中的半衰期通常不足，因此患有慢性疾病的患者需要经常接受给药，通常是通过痛苦的注射。这些繁重的给药方案导致患者不依从率高，伴随着不良反应和负面的健康结果。具有改善的血浆半衰期的治疗性蛋白质将允许放宽给药时间表，降低给药成本并通过减少不依从性来改善结果。这项拟议的技术专注于改善主要影响儿童的慢性疾病治疗剂的血浆半衰期，目前全球市场规模为40亿美元。类似地，工业酶经常部署在损害酶稳定性和活性的恶劣环境中。赋予酶对破坏稳定的化学物质的抵抗力，将使它们能够在目前禁止的高价值环境中部署。该技术将用于稳定酶，以应用于价值7.3亿美元的个性化医疗市场。如果成功，这项工作将为开发可在目前禁止的环境中部署的下一代酶提供平台。SBIR第一阶段项目的智力价值是利用合成生物学平台来创建具有新氨基酸构建块的蛋白质，从而显着提高半衰期和稳定性。许多用作治疗剂或用作工业酶的蛋白质通过二硫键稳定。这些键在称为还原剂的化学物质的存在下断裂，这些化学物质存在于人血浆中（对治疗剂不稳定）以及溶剂和缓冲液中（对工业酶不稳定）。利用工程E.大肠杆菌可以将超过20种标准氨基酸的氨基酸整合到蛋白质中，目标是用形成称为二硒醚的键的硒代半胱氨酸氨基酸取代形成二硫化物的半胱氨酸氨基酸。二硒化物稳定的蛋白质在具有与二硫化物稳定的蛋白质不相容的还原剂的环境中保持稳定性和活性。该项目将生产一种用于主要疾病类别的二硒化物稳定蛋白质治疗剂。在长时间暴露于血浆后，将通过显示改善的结合活性的ELISA测定和显示改善的治疗活性的基于细胞的测定来证明对二硫化物稳定的治疗剂的改善。该项目还将生产一种二硒稳定的工业酶，以催化个性化医疗的关键反应。通过还原条件下的体外稳定性和活性测定，将证明二硫化物稳定酶的性能改进。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to develop technology using engineered bacteria to improve protein stability in two large markets: therapeutic proteins and industrial enzymes. Proteins used as therapeutics frequently have insufficient half-lives in human blood plasma, so that patients with chronic disease need to receive frequent dosings, often by painful injections. These burdensome dosing schedules result in high rates of patient noncompliance, with attendant poor responses and negative health outcomes. Therapeutic proteins with improved half-lives in blood plasma would permit relaxed dosing schedules, lowering costs of administration and improving outcomes by reducing noncompliance. This proposed technology focuses on improving the plasma half-life of a therapeutic for a chronic disease primarily affecting children that currently comprises a $4B global market. Similarly, industrial enzymes are frequently deployed in harsh environments that impair enzyme stability and activity. Endowing enzymes with improved resistance to destabilizing chemicals would enable their deployment in high-value environments that are currently prohibitive. The proposed technology will be used to stabilize an enzyme for application in a $730M segment of the personalized medicine market. If successful, this work would provide a platform for developing next-generation enzymes deployable in currently prohibitive environments. The intellectual merit of this SBIR Phase I project is to utilize a synthetic biology platform to create proteins with new amino acid building blocks that dramatically improve half-life and stability. Many proteins used as therapeutics or used as industrial enzymes are stabilized by disulfide bonds. These bonds break in the presence of chemicals called reducing agents that are found both in human blood plasma (destabilizing to therapeutics) and in solvents and buffers (destabilizing to industrial enzymes). Using a strain of engineered E. coli that can incorporate amino acids beyond the 20 standard amino acids into proteins, the goal is to replace disulfide-forming cysteine amino acids with selenocysteine amino acids that form bonds called diselenides. Diselenide-stabilized proteins maintain stability and activity in environments with reducing agents incompatible with disulfide-stabilized proteins. This project will produce a diselenide-stabilized protein therapeutic for a major disease class. Improvements to disulfide-stabilized therapeutics will be demonstrated by ELISA assays showing improved binding activity, and by cell-based assays showing improved therapeutic activity, after prolonged exposure to blood plasma. This project also will produce a diselenide-stabilized industrial enzyme to catalyze a critical reaction for personalized medicine. Performance improvements over disulfide-stabilized enzymes will be demonstrated by in vitro stability and activity assays in reducing conditions.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.