Developing a protein microarray technology for high throughput proteomic research

描述（由申请人提供）：蛋白质微阵列技术具有高样本密度和多重实验设置，是在整个蛋白质组水平研究系统生物学的有力工具。蛋白质微阵列技术在生物医学研究和药物开发中具有广泛的用途。该技术最突出的应用是疾病生物标志物发现和体外诊断。这项技术的成功非常符合NIH的使命，即改善公众健康，开发新的方法和技术来解决未满足的医疗需求。尽管DNA微阵列的发展为高密度微阵列的制备提供了技术平台，但组装蛋白质微阵列的最大挑战仍然是获得包含整个人类蛋白质组的高质量功能蛋白质。目前最全面的蛋白质微阵列芯片仅包含人类蛋白质组的三分之一，而且该芯片上的蛋白质是在昆虫细胞表达系统中产生的，缺乏适当的哺乳动物翻译后修饰。蛋白质微阵列技术应用的主要障碍是缺乏高质量的商业化芯片。OriGene Technologies，Inc.是一家以基因为中心的生物技术公司。为了引领人类重组蛋白生产市场，OriGene将超过15，000个ORF cDNA穿梭到其专有的真核表达系统中，并在源自人类细胞的HEK 293 T细胞系中产生12，000个C末端myc和Flag表位标记的人类蛋白质，并通过抗Flag蛋白质印迹分析验证其表达。这项前所未有的工作使OriGene处于令人羡慕的位置，可以生成最好的人类蛋白质微阵列芯片。我们广泛的前期工作也表明，我们将能够为研究试剂市场生产最高质量的蛋白质微阵列芯片。在第一阶段的申请中，我们希望开发一种原型高密度蛋白质微阵列芯片，其中包含HEK 293 T细胞中产生的10，464种独特的重组人类蛋白质。该蛋白质芯片的实用性将通过检测蛋白质-蛋白质相互作用来证明。该项目的预期期限为一年。该赠款的第二阶段将扩展到这种蛋白质芯片技术的商业化，并生产一种高度纯化的蛋白质微阵列芯片，其中至少含有20，000种独特的人类蛋白质。还将开发和优化多种实用程序。我们相信，这种高密度蛋白质微阵列芯片技术将成为生物医学研究以及药物研发的颠覆性技术。 公共卫生相关性：蛋白质微阵列技术在生物医学研究和药物开发中具有广泛的用途，包括疾病生物标志物的发现和体外诊断。这项技术的发展非常符合NIH的使命，即改善公众健康，开发新的方法和技术来解决未满足的医疗需求。

DESCRIPTION (provided by applicant): With its high sample density and multiplexed experimental settings, protein microarray technology is a powerful tool to study system biology at the whole proteome level. Protein microarray technology has a broad-range of utilities in biomedical research and pharmaceutical development. The most prominent application for this technology is disease biomarker discovery and in vitro diagnosis. The success of this technology fits well with NIH's mission for improving public health and developing novel methods and technologies to address unmet medical needs. Although the development of DNA microarrays has provided the technological platform for high-density microarray fabrication, the greatest challenge in assembling protein microarrays is still the acquisition of high-quality functional proteins encompassing the entire human proteome. The most comprehensive protein microarray chip currently available only includes one third of the human proteome, and furthermore, the proteins on this chip were produced in an insect cell expression system and lack appropriate mammalian posttranslational modifications. The major impediment to the application of protein microarray technology is the lack of a high-quality commercially available chip. OriGene Technologies, Inc. is a gene centric biotech company. To spearhead the human recombinant protein production market, OriGene shuttled more than 15,000 ORF cDNAs into its proprietary Eukaryotic expression system and produced 12,000 C-terminal myc- and Flag-epitope tagged human proteins in a human cell originated HEK293T cell line and validated their expression via an anti-Flag Western blot analysis. This unprecedented work has put OriGene in an enviable position to generate the best human protein microarray chip. Our extensive preliminary work also demonstrated that we will be able to generate the highest quality protein microarray chips for the research reagent market. In this Phase I application, we expect to develop a prototype high density protein microarray chip that contains 10,464 unique recombinant human proteins produced in HEK293T cells. The utilities of this protein chip will be demonstrated by detection of protein-protein interactions. The expected duration of the project is one year. Phase II of the grant will be an expansion into the commercialization of this protein chip technology, and the production of a highly purified protein microarray chip spotted with at least 20,000 unique human proteins. Multiple utilities will also be developed and optimized. We believe that this high density protein microarray chip technology will become a disruptive technology for biomedical research, as well as pharmaceutical research and development. PUBLIC HEALTH RELEVANCE: Protein microarray technology has a broad-range of utilities in biomedical research and pharmaceutical development, including disease biomarker discovery and in vitro diagnosis. The development of this technology fits well with NIH's mission for improving public health and developing novel methods and technologies to address unmet medical needs.