Proteases and tags for protein purification and analysis

描述(由申请人提供)：长期目标是将一种从克隆基因中快速纯化蛋白质的系统商业化。该系统既适用于实验室规模的高通量蛋白质的并行纯化，也适用于药用蛋白质的过程规模的纯化。这项技术是美国国立卫生研究院资助的关于枯草杆菌蛋白酶枯草杆菌前结构域介导的折叠反应研究的结果。这个系统的两个基本组成部分是：1)高度工程化的蛋白酶(PSUB)，它在氟化物触发下对特定底物进行水解性反应；2)PSUB与其原结构域的工程化版本(PROTAG)之间的高亲和力相互作用。这些组分结合在一起形成了一个纯化系统，其中固定化的PSUB既用作亲和纯化标记融合蛋白的结合分子，又用作去除PROTAG的加工酶。这四个实验目标是：1)工程定点固定化PSUB；2)优化PSUB的规模化生产和纯化；3)建立和测试高通量的蛋白质纯化、定量和分析方法；4)鉴定具有精细化学触发的第二代psubs。重组蛋白经常与其他蛋白质或多肽融合，以便于表达和纯化。这些标签为亲和纯化提供了一个临时的挂钩，但最终必须由一种特定于位点的蛋白酶处理。然而，标签移除通常是低效的，有时还会出现问题。该系统的技术创新是将标签去除整合到净化过程中。这提供了任何其他系统所不具备的简单性和效率。这项技术应该会让任何提纯蛋白质的人受益，但它对依赖于并行处理样本的结构基因组学努力的影响应该特别大。这项技术最终将有利于药用蛋白质的规模化提纯。快速产生大量治疗性蛋白的能力对于应对自然出现的和故意引入的病原体都可能是至关重要的。这个项目中正在开发的技术应该会使任何纯化蛋白质的人受益，但对依赖于高通量、并行处理样本的结构基因组学努力的影响应该特别大。这项技术最终将有利于药用蛋白质的大规模纯化。快速产生大量治疗性蛋白的能力对于应对自然出现的和故意引入的病原体都可能是至关重要的。

DESCRIPTION (provided by applicant): The long-term objective is to commercialize a system for rapid purification of proteins from cloned genes. The system should be suitable for both high throughput, parallel purification of proteins on the laboratory scale, as well as process-scale purification of pharmaceutical proteins. This technology was developed as a result of NIH-funded studies on the prodomain- mediated folding reaction of the Bacillus protease subtilisin. The two fundamental components of this system are: 1) A highly engineered protease (psub) which hydrolyzes specific substrates in response to a fluoride trigger; 2) The high affinity interaction between psub and an engineered version of its prodomain (protag). This components are combined to create a purification system in which immobilized psub is used both as the binding molecule for affinity purification of protagged fusion proteins and as the processing protease for protag removal. The four experimental aims are: 1) Engineer site-directed immobilization of psub; 2) Optimize large scale production and purification of psub; 3) Create and test high-throughput methods for protein purification, quantitation and analysis; 4) Identify second generation psubs with refined chemical triggers. Recombinant proteins are frequently fused with other proteins or peptides to facilitate expression and purification. The tags provide a temporary hook for affinity purification, but ultimately must be processed by a site-specific protease. Tag removal, however, is frequently inefficient and sometimes problematic. The technical innovation of the system is the integration of tag removal into the purification process. This provides simplicity and efficiency that isn't available in any other system. The technology should benefit anyone purifying proteins but the impact on structural genomics efforts which rely on parallel processing of samples should be particularly great. The technology should eventually benefit process scale purification of pharmaceutical proteins. The ability to rapidly produce large quantities of therapeutic proteins could be critical for responding to both naturally-emergent and intentionally-introduced pathogens. The technology being developed in this project should benefit anyone purifying proteins but the impact on structural genomics efforts which rely on high throughput, parallel processing of samples should be particularly great. The technology should eventually benefit large-scale purification of pharmaceutical proteins. The ability to rapidly produce large quantities of therapeutic proteins could be critical for responding to both naturally-emergent and intentionally-introduced pathogens.