Toxoid adjuvant CRM197 production in a stable reduced genome E. coli strain

描述（由申请人提供）：Scarab Genomics开发了有价值的大肠杆菌基因组减少菌株，其中多个缺失从基因组中删除了许多不需要的和所有潜在危险的DNA。这些Clean Genome（R）菌株，沿着Scarab的基因表达技术，提供了一个非常有效地生产高产量优质蛋白质的系统。在这个I期项目中，该系统的优点将被应用于称为CRM 197的白喉类毒素的生产。这种蛋白质通过活性位点的突变而变得无毒，但仍然具有高度抗原性。它形成了佐剂部分 目前用于保护婴儿和儿童免受细菌性脑膜炎的结合疫苗。CRM197很难大量生产，因此非常昂贵。如果这些疫苗和其他结合疫苗能够更有效地生产，它们就可以被更广泛的人群所接受。 使用从金龟子收集物中选择的菌株，我们将鉴定宿主和载体的最佳组合以表达CRM197。一组有用的突变和缺失的多种组合是可用的。这些宿主菌株将与携带严格控制的诱导型启动子的质粒组合用于表达测试，以驱动CRM 197蛋白质产生。该表达质粒可用于任何E.大肠杆菌菌株，因此可以直接比较不同Clean Genome（R）宿主的性能。还将评估不同的表达方式。为了产生可溶性CRM 197，将使用信号引导分泌到内膜和外膜之间的周质空间中。我们最近的工作表明了实现高水平周质生产的新策略。或者，在细胞质中以高水平产生的CRM 197将经受当代重折叠方法以再生可溶形式。这是一种可行的生产策略，已用于生产许多生物药物。这些实验将表明宿主菌株和表达模式的最佳组合。最后，Scarab的优化方案将被应用于确定易于纯化的产品的最高收率的条件。 第一阶段项目应确定改进这一重要蛋白质生产工艺的可行性。如果成功的话，这一系统将使圣甲虫能够利用迅速扩大的市场，其目的是改善弱势群体获得所需疫苗的机会。 公共卫生相关性：预期Clean Genome（R）表达系统将提高生产当前使用的缀合疫苗的重要蛋白质组分的效率并降低其成本。这种蛋白质也将比具有非减少基因组的传统细菌产生的蛋白质更清洁和安全。这些改进可能对药物蛋白质产品的生产产生广泛影响，并最终扩大需要疫苗的高危人群获得疫苗的机会。

DESCRIPTION (provided by applicant): Scarab Genomics has developed valuable reduced-genome strains of Escherichia coli in which multiple deletions remove from the genome much unwanted and all potentially hazardous DNA. These Clean Genome(R) strains, along with Scarab's gene expression technology, provide a system for producing high yields of premium-quality proteins very efficiently. In this Phase I project the advantages of the system will be applied to production of a diphtheria toxoid known as CRM197. This protein is rendered non-toxic by a mutation in the active site, but remains highly antigenic. It forms the adjuvant portion in conjugate vaccines currently used to protect infants and children against bacterial meningitis. CRM197 is difficult to manufacture in quantity and therefore very expensive. These and other conjugate vaccines could become within the reach of wider populations if they could be manufactured much more efficiently. Using strains selected from Scarab's collection we will identify the optimal combination of host and vector to express CRM197. A panel of multiple combinations of useful mutations and deletions are available. These host strains will be used for expression tests in combination with a plasmid bearing a tightly controlled inducible promoter to drive CRM197 protein production. The expression plasmid can be used in any E. coli strain, so performance of different Clean Genome(R) hosts can be directly compared. Different modes of expression will also be evaluated. To produce soluble CRM197 signal-directed secretion into the periplasmic space between the inner and outer membranes will be used. Our recent work indicates new strategies to achieve high levels of periplasmic production. Alternatively, CRM197 produced at high levels in the cytoplasm, will be subjected to contemporary refolding methods to regenerate the soluble form. This is a feasible production strategy that has been used to produce a number of biopharmaceuticals. These experiments will indicate the best combination of host strain and expression mode. Finally, Scarab's optimization protocol will be applied to determine conditions for the highest yield of easily purifiable product. The Phase I project should establish the feasibility of an improved production process for this important protein. If successful, this system could enable Scarab to take advantage of a rapidly expanding market with a process aimed at improving access of vulnerable populations to needed vaccines. PUBLIC HEALTH RELEVANCE: The Clean Genome(R) expression system is expected to increase the efficiency and reduce the cost of manufacturing an important protein component of conjugate vaccines in current use. The protein will also be cleaner and safer than that produced in conventional bacteria with non-reduced genomes. These improvements could have a wide impact on production of pharmaceutical protein products and ultimately broaden access to vaccines for at-risk populations who need them.