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/商业潜力是开发利用工程细菌的技术，以提高两大市场的蛋白质稳定性：治疗蛋白质和工业酶。用作治疗药物的蛋白质在人血浆中的半衰期往往不够，因此患有慢性疾病的患者需要频繁给药，通常是痛苦的注射。这些繁重的给药计划导致患者不遵守规定的比例很高，随之而来的是不良反应和负面的健康结果。改善血浆半衰期的治疗蛋白将允许放宽给药计划，降低给药成本，并通过减少不遵医嘱而改善结果。这项拟议的技术专注于改善一种主要影响儿童的慢性疾病的治疗药物的血浆半衰期，目前该药物的全球市场规模为40亿美元。同样，工业酶经常被部署在破坏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抗性，将使它们能够在目前禁止的高价值环境中部署。这项拟议中的技术将用于稳定一种酶，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当前令人望而却步的环境中部署的下一代酶提供一个平台。这个SBIR一期项目的智力优势在于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。许多用作治疗药物或用作工业酶的蛋白质是由二硫键稳定的。这些化学键在被称为还原剂的化学物质存在时断裂，这种还原剂既存在于人体血浆中（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结合到蛋白质中的工程大肠杆菌菌株，目标是用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，这种氨基酸形成一种叫做二硒化半胱氨酸的键。二硫化物稳定的蛋白质在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治疗一类主要疾病的双硒稳定蛋白。在长期暴露于血浆后，将通过ELISA检测显示结合活性的改善，以及基于细胞的检测显示治疗活性的改善来证明对二硫化物稳定疗法的改进。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。在还原条件下，通过体外稳定性和活性测定，可以证明二硫稳定酶的性能改进。该奖项反映了美国国家科学基金会的法定使命，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.