High-throughput protein expression of cDNA products as a tool in functional genomics

A proteomics approach has been developed aimed to allow high throughput ana lysis 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 th e proteins on cellular and subcellular level. To evaluate the system, 55 cD NA clones with predominantly unknown function were selected from a mouse te stis cDNA-library. A bacterial expression system was designed that allowed robust expression and easy purification. Protein levels between 15 and 80 m g l(-1) were obtained for 49 of the clones. Five clones were selected for i mmunization and all yielded functional antibodies that gave specific staini ng in Western blot screening of samples from various cell types. Furthermor e, extensive immunolocalization information on subcellular level was obtain ed for three of the five clones. All generated data were stored in a relati onal database, and are made available through a web-interface (http://www.b iochem.kth.se/multiscale/), which also provides relevant links and allows h omology searches from the original sequences. The possibility to allow anal ysis of gene products from whole genomes using this 'localization proteomic s' approach is discussed. (C) 2000 Published by Elsevier Science B.V. All r ights reserved.