Nanoliter Lab-on-a-chip for Protein Crystallization

描述(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，可能产生100,000个多肽产品。这些产物组装成100多万个生物相关结构或每个有机体感兴趣的蛋白质。这些蛋白质的结晶和X射线衍射是常规进行的，以确定它们的3D结构，这是任何有效的蛋白质工程、合理的药物设计或受控药物输送所必需的。蛋白质的晶体结构，如酶、核糖体蛋白、细菌毒素、激素和受体，对于理解重要的生理过程以及理解和治疗微生物引起的疾病是必不可少的。最近的抗艾滋病药物都是酶抑制剂，它们的设计是基于酶的晶体结构提供的详细的蛋白质-药物相互作用，基于结构-活性关系。 为了获得蛋白质晶体，需要对大量的蛋白质进行筛选，以找到有利于结晶的条件，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。在天然蛋白质需要结晶的情况下，它们的供应量可能非常有限。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，我们成功地展示了一种使用溶菌酶、葡萄糖异构酶和蛋白酶K等3种蛋白质的25纳升蛋白质液滴的芯片上蛋白质结晶实验室。每个筛选条件的蛋白质量如此之少，大大节省了上游蛋白质表达和纯化的成本。在第一阶段，我们解决了多个研究问题，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，我们将在这些研究成果的基础上，开发只需一滴蛋白质就能筛选出384种粗网格试剂的筛选芯片，并开发一种优化芯片，它将可编程地从仅5个盐、沉淀剂、缓冲液、水和蛋白质的储存库中自动在芯片上设置96个精细网格条件。我们的仪器和一次性芯片即使对个人调查员来说也是负担得起的。 蛋白质的晶体结构，如酶、核糖体蛋白、细菌毒素、激素和受体，对于理解重要的生理过程是必不可少的。这些结构信息将有助于揭示蛋白质在健康和疾病中扮演的角色，并有助于合理设计新药。最近的抗艾滋病药物都是酶抑制剂，它们的设计是基于酶的晶体结构提供的详细的蛋白质-药物相互作用，基于结构-活性关系。

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.