Two-dimensional gel electrophoresis can resolve virtually all of the individual proteins of a living cell, making construction of a total protein database for a given cell a realistic goal. Such a database should trace each protein to its structural gene and account for every structural gene of the cell. We will continue construction of a two-dimensional map of the proteins encoded by the bacterium Escherichia coli using gel electrophoresis in two new approaches. In the first, the novel transcription system of phage T7 will be used to express the genes on an ordered set of cloned chromosomal DNA segments spanning the E. coli genome (the Kohara library). The cloned segments will be individually spliced into an expression vector containing the T7 promoter, and expressed in a cell containing an IPTG-inducible T7 RNA polymerase gene. The coordinates marking the location of the expressed radioactive proteins in a standard gel system will be recorded. The second, complementary approach will utilize the increasing amount of DNA sequence information for E. coli to identify putative genes as ORFs (open reading frames) and to predict the location on gels of their potential polypeptide products, with amino acid composition helping verify the identification. The data gathered by these new approaches will be combined with the current E. coli gene-protein index and assembled in electronic form for integration with the overall database being developed by a consortium of E. coli laboratories.
