Nanoliter Lab-on-a-chip for Protein Crystallization

用于蛋白质结晶的纳升芯片实验室

• 批准号: 7155454
• 负责人: VAMSEE K. PAMULA
• 金额: $ 98.76万
• 依托单位: ADVANCED LIQUID LOGIC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155454
• 关键词: automated data processing; bioengineering /biomedical engineering; biomedical equipment development; chemical standardization; consumable /disposable biomedical equipment; crystallization; genetic screening; microarray technology; microfluidics; microprocessor /microchip; nanotechnology; protein structure function; reagent /indicator

DESCRIPTION (provided by applicant): The human genome contains at least 30,000 unique open reading frames that may yield >100,000 polypeptide products. These products assemble into more than a million biologically relevant structures or proteins of interest per organism. Crystallization and X-ray diffraction of these proteins is routinely performed to determine their 3D structure which needs to be understood for any effective protein engineering, rational drug design, or controlled drug delivery. Crystal structures of proteins such as enzymes, ribosomal proteins, bacterial toxins, hormones, and receptors are essential to understand important physiological processes and also to understand and treat diseases caused by microorganisms. Recent antiviral drugs against AIDS are enzyme inhibitors, and their design was based on the detailed protein-drug interactions provided by crystal structures of the enzymes with the drugs, based on structure-activity relationships. In order to obtain a protein crystal, large amounts of protein need to be screened to find favorable conditions for crystallization but unfortunately many interesting proteins are very expensive or sometimes even difficult to express in large quantities. And in cases where native proteins are desirable to be crystallized, they could be available in extremely limited quantities. The equipment currently used for screening and optimization of crystallization conditions is very expensive and uses large amounts of precious proteins. In phase I, we have successfully demonstrated a protein crystallization lab-on-a-chip using 25 nanoliter protein droplets of 3 proteins viz., lysozyme, glucose isomerase, and proteinase K. Such low volumes of protein per screening condition translate to significant cost savings in the upstream protein expression and purification. During phase I, we resolved a number of research issues to demonstrate the feasibility of performing crystallization screening on a digital microfluidic chip while in Phase II we will build on those results to: develop a screening chip that requires only 1 drop of protein to screen against 384 coarse grid reagents and develop an optimization chip which will programmably and automatically set up 96 fine grid conditions on-chip from just 5 reservoirs of salt, precipitant, buffer, water, and protein. Our instrument and disposable chips would be affordable even for an individual investigator. Crystal structures of proteins such as enzymes, ribosomal proteins, bacterial toxins, hormones, and receptors are essential to understand important physiological processes. This structural information will help reveal the roles that proteins play in health and disease and will help rational design of new medicines. Recent antiviral drugs against AIDS are enzyme inhibitors, and their design was based on the detailed protein-drug interactions provided by crystal structures of the enzymes with the drugs, based on structure-activity relationships.

描述（由申请人提供）：人类基因组包含至少 30,000 个独特的开放阅读框，可产生 >100,000 种多肽产物。这些产品组装成每个生物体超过一百万个生物学相关结构或感兴趣的蛋白质。通常对这些蛋白质进行结晶和 X 射线衍射以确定它们的 3D 结构，这需要了解任何有效的蛋白质工程、合理的药物设计或受控药物递送。酶、核糖体蛋白、细菌毒素、激素和受体等蛋白质的晶体结构对于了解重要的生理过程以及了解和治疗微生物引起的疾病至关重要。最近针对艾滋病的抗病毒药物是酶抑制剂，其设计基于酶与药物的晶体结构所提供的详细的蛋白质-药物相互作用，并基于构效关系。 为了获得蛋白质晶体，需要筛选大量蛋白质以找到有利的结晶条件，但不幸的是许多有趣的蛋白质非常昂贵，有时甚至难以大量表达。在需要结晶天然蛋白质的情况下，它们的数量可能极其有限。目前用于筛选和优化结晶条件的设备非常昂贵，并且使用大量珍贵的蛋白质。 在第一阶段，我们成功地展示了使用 3 种蛋白质（即溶菌酶、葡萄糖异构酶和蛋白酶 K）的 25 纳升蛋白质液滴的蛋白质结晶实验室。每个筛选条件的蛋白质体积如此之低意味着巨大的成本节省上游蛋白质表达和纯化。在第一阶段，我们解决了许多研究问题，以证明在数字微流控芯片上进行结晶筛选的可行性，而在第二阶段，我们将在这些结果的基础上：开发一种仅需要 1 滴蛋白质即可筛选的筛选芯片384 个粗网格试剂并开发了一个优化芯片，该芯片将通过 5 个盐、沉淀剂、缓冲液、水和蛋白质储存器，以可编程方式自动在芯片上设置 96 个精细网格条件。即使对于个体研究者来说，我们的仪器和一次性芯片也是负担得起的。 酶、核糖体蛋白、细菌毒素、激素和受体等蛋白质的晶体结构对于理解重要的生理过程至关重要。这些结构信息将有助于揭示蛋白质在健康和疾病中发挥的作用，并有助于合理设计新药物。最近针对艾滋病的抗病毒药物是酶抑制剂，其设计基于酶与药物的晶体结构所提供的详细的蛋白质-药物相互作用，基于结构-活性关系。