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）：人类基因组至少包含30,000个独特的开放式阅读框架，可能产生> 100,000个多肽产品。这些产品将每个生物体的生物学相关结构或感兴趣的蛋白质组装成超过一百万个。通常执行这些蛋白质的结晶和X射线衍射，以确定其3D结构，对于任何有效的蛋白质工程，合理的药物设计或受控药物递送，都需要理解其3D结构。蛋白质的晶体结构，例如酶，核糖体蛋白质，细菌毒素，激素和受体对于了解重要的生理过程至关重要，也可以理解和治疗由微生物引起的疾病。最近针对艾滋病的抗病毒药物是酶抑制剂，其设计基于基于结构 - 活性关系，由酶的晶体结构与药物的晶体结构提供的详细蛋白质 - 药物相互作用。 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，有时甚至很难大量表达。如果需要结晶的天然蛋白质，则可能会以极限的量提供。当前用于筛选和优化结晶条件的设备非常昂贵，并且使用了大量珍贵蛋白质。 在第一阶段，我们成功地证明了使用3种蛋白质的25个纳米蛋白蛋白液滴，即3种蛋白质，溶菌酶，葡萄糖异构酶和蛋白酶K。每次筛选条件的这种低体积的蛋白质转化为上Upstream蛋白质表达和纯化的大量储蓄。在第一阶段，我们解决了许多研究问题，以证明在数字微流体芯片上执行结晶筛选的可行性，而在第二阶段，我们将基于这些结果，以：开发筛选芯片，仅需要1滴蛋白质才能筛选筛选384个粗网格试剂，并开发出584个粗网格和自动设置96固定条件的壳，并在96上进行了自动设置，并将其定为96-ensot On-in-in-in-in-in cons，并自动设置了96次固定条件。沉淀物，缓冲液，水和蛋白质。即使对于个人调查员来说，我们的仪器和一次性芯片也将负担得起。 蛋白质的晶体结构，例如酶，核糖体蛋白质，细菌毒素，激素和受体对于了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起起作用的作用，并有助于新药物的合理设计。最近针对艾滋病的抗病毒药物是酶抑制剂，其设计基于基于结构 - 活性关系，由酶的晶体结构与药物的晶体结构提供的详细蛋白质 - 药物相互作用。