Larsson M, Gräslund S, Yuan L, Brundell E, Uhlén M, Höög C, Ståhl S
Department of Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden.
J Biotechnol. 2000 Jun 23;80(2):143-57. doi: 10.1016/s0168-1656(00)00258-3.
A proteomics approach has been developed aimed to allow high throughput analysis of protein products expressed from cDNA fragments (expressed sequence tags, ESTs). The concept relies on expression of gene products to generate specific antibodies for protein analysis, such as immunolocalization of the proteins on cellular and subcellular level. To evaluate the system, 55 cDNA clones with predominantly unknown function were selected from a mouse testis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 mg l(-1) were obtained for 49 of the clones. Five clones were selected for immunization and all yielded functional antibodies that gave specific staining in Western blot screening of samples from various cell types. Furthermore, extensive immunolocalization information on subcellular level was obtained for three of the five clones. All generated data were stored in a relational database, and are made available through a web-interface (http://www.biochem.kth.se/multiscale/), which also provides relevant links and allows homology searches from the original sequences. The possibility to allow analysis of gene products from whole genomes using this 'localization proteomics' approach is discussed.
已开发出一种蛋白质组学方法，旨在对从cDNA片段（表达序列标签，ESTs）表达的蛋白质产物进行高通量分析。该概念依赖于基因产物的表达来生成用于蛋白质分析的特异性抗体，例如在细胞和亚细胞水平上对蛋白质进行免疫定位。为了评估该系统，从小鼠睾丸cDNA文库中选择了55个功能主要未知的cDNA克隆。设计了一种细菌表达系统，该系统能够实现高效表达且易于纯化。49个克隆的蛋白质产量在15至80 mg l(-1)之间。选择了5个克隆进行免疫，所有克隆均产生了功能性抗体，这些抗体在对来自各种细胞类型的样品进行蛋白质印迹筛选时呈现特异性染色。此外，还获得了5个克隆中的3个在亚细胞水平上的广泛免疫定位信息。所有生成的数据都存储在关系数据库中，并通过网络界面（http://www.biochem.kth.se/multiscale/）提供，该界面还提供相关链接，并允许从原始序列进行同源性搜索。讨论了使用这种“定位蛋白质组学”方法分析来自全基因组的基因产物的可能性。
