Proteases and tags for protein purification and analysis

描述（由申请人提供）：长期目标是将用于从克隆基因中快速纯化蛋白质的系统商业化。该系统应适用于实验室规模的蛋白质的高通量平行纯化以及药物蛋白质的工艺规模纯化。该技术是NIH资助的关于芽孢杆菌蛋白酶枯草杆菌蛋白酶的前结构域介导的折叠反应的研究的结果。该系统的两个基本组成部分是：1）高度工程化的蛋白酶（psub），其响应于氟化物触发剂而水解特定底物; 2）psub与其前结构域的工程化版本（protag）之间的高亲和力相互作用。将这些组分组合以产生纯化系统，其中固定化的psub既用作亲和纯化前标签融合蛋白的结合分子，又用作去除前标签的加工蛋白酶。四个实验目标是：1）工程定点固定psub; 2）优化psub的大规模生产和纯化; 3）创建和测试用于蛋白质纯化、定量和分析的高通量方法; 4）鉴定具有精制化学触发物的第二代psub。重组蛋白经常与其他蛋白或肽融合以促进表达和纯化。标签为亲和纯化提供了一个临时的钩子，但最终必须由位点特异性蛋白酶处理。然而，标签移除通常是低效的，并且有时是有问题的。该系统的技术创新是将标签去除集成到净化过程中。这提供了任何其他系统都无法提供的简单性和效率。这项技术应该对任何纯化蛋白质的人都有好处，但对依赖于平行处理样品的结构基因组学工作的影响应该特别大。该技术最终将有利于药物蛋白质的工艺规模纯化。快速产生大量治疗性蛋白质的能力对于应对自然出现的和有意引入的病原体都至关重要。该项目正在开发的技术应该使任何纯化蛋白质的人受益，但对依赖于高通量，平行处理样品的结构基因组学工作的影响应该特别大。该技术最终将有利于大规模纯化药用蛋白质。快速产生大量治疗性蛋白质的能力对于应对自然出现的和有意引入的病原体都至关重要。

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.