Vinarov Dmitriy A, Markley John L
Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, WI 53706-1549, USA.
Expert Rev Proteomics. 2005 Jan;2(1):49-55. doi: 10.1586/14789450.2.1.49.
The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein or protein fragment on a preparative scale is one of the most important tasks in biotechnology today. The Center for Eukaryotic Structural Genomics (WI, USA), in co-operation with Ehime University (Matsuyama, Japan) and CellFree Sciences Co., Ltd, has developed an automated platform for nuclear magnetic resonance-based structural proteomics that employs wheat germ extracts for cell-free production of labeled protein. The platform utilizes a single construct for all targets without any redesign of the DNA or RNA. Therefore, it offers advantages over commercial cell-free methods utilizing Escherichia coli extracts that require multiple constructs or redesign of the open reading frame. The protein production and labeling protocol is no more costly than E. coli cell-based approaches, is robust and scalable for high-throughput applications. This protocol has been used in the authors center to screen eukaryotic open reading frames from the Arabidopsis thaliana and human genomes and for the determination of nuclear magnetic resonance structures. With the recent addition of the GeneDecoder 1000 (CellFree Sciences Co., Ltd) robotic system, the Center for Eukaryotic Structural Genomics is able to carry out as many as 384 small-scale (50 microl) screening reactions per week. Furthermore, the Protemist (CellFree Sciences Co., Ltd) robotic system enables the Center for Eukaryotic Structural Genomics to carry out 16 production-scale (4 ml) reactions per week. Utilization of this automated platform technology to screen targets for expression and solubility and to produce stable isotope-labeled samples for nuclear magnetic resonance structure determinations is discussed.
开发能够在制备规模上合成任何所需的可溶性、标记蛋白质或蛋白质片段的新系统和策略是当今生物技术中最重要的任务之一。美国威斯康星州的真核结构基因组学中心与日本松山的爱媛大学以及无细胞科学有限公司合作，开发了一个基于核磁共振的结构蛋白质组学自动化平台，该平台利用小麦胚芽提取物进行无细胞标记蛋白质生产。该平台对所有目标使用单一构建体，无需对DNA或RNA进行任何重新设计。因此，与利用大肠杆菌提取物的商业无细胞方法相比，它具有优势，后者需要多个构建体或对开放阅读框进行重新设计。蛋白质生产和标记方案的成本并不高于基于大肠杆菌细胞的方法，对于高通量应用而言稳健且可扩展。该方案已在作者所在的中心用于筛选拟南芥和人类基因组中的真核开放阅读框以及用于核磁共振结构的测定。随着最近添加了GeneDecoder 1000（无细胞科学有限公司）机器人系统，真核结构基因组学中心每周能够进行多达384次小规模（50微升）筛选反应。此外，Protemist（无细胞科学有限公司）机器人系统使真核结构基因组学中心每周能够进行16次生产规模（4毫升）反应。本文讨论了利用这种自动化平台技术筛选目标以进行表达和溶解性分析以及生产用于核磁共振结构测定的稳定同位素标记样品的情况。
