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

这个小型企业创新研究(SBIR)第一阶段项目的更广泛影响/商业潜力是开发使用工程菌的技术，以提高两大市场的蛋白质稳定性：治疗性蛋白质和工业酶。用作治疗的蛋白质在人体血浆中的半衰期经常不足，因此慢性病患者需要经常接受剂量，通常是通过痛苦的注射。这些繁重的剂量计划导致患者不依从性的高比率，随之而来的是不良反应和负面的健康结果。血浆中半衰期改善的治疗性蛋白质将允许放松给药计划，降低给药成本，并通过减少不依从性来改善结果。这项拟议技术的重点是改善一种主要影响儿童的慢性疾病治疗药物的血浆半衰期，目前全球市场规模为40亿美元。同样，工业酶经常被部署在恶劣的环境中，这会损害酶的稳定性和活性。赋予酶对破坏稳定的化学品更好的抵抗力，将使它们能够部署在目前令人望而却步的高价值环境中。这项拟议的技术将用于稳定一种酶，以便在7.3亿美元的个性化药物市场中应用。如果成功，这项工作将为开发下一代酶提供一个平台，可以在目前令人望而却步的环境中部署。SBIR第一阶段项目的智力优势是利用合成生物学平台创造具有新氨基酸构建块的蛋白质，显著改善半衰期和稳定性。许多用作治疗或工业酶的蛋白质是通过二硫键稳定的。这些键在被称为还原剂的化学物质的存在下断裂，这种化学物质既存在于人类血浆中(对治疗药物不稳定)，也存在于溶剂和缓冲液中(对工业酶不稳定)。利用一种可以将20种标准氨基酸以外的氨基酸整合到蛋白质中的工程大肠杆菌，其目标是用形成二硫键的硒半胱氨酸氨基酸取代形成二硫键的半胱氨酸氨基酸，这种氨基酸被称为二硒醚。二硒醚稳定的蛋白质在含有与二硫键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治疗一种重大疾病的双硒醚稳定蛋白。二硫化物稳定疗法的改进将通过显示结合活性提高的ELISA法和长期暴露于血浆后显示改善治疗活性的基于细胞的分析来证明。该项目还将生产一种二硒醚稳定的工业酶，以催化个性化药物的关键反应。在减少条件下的体外稳定性和活性分析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 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.