'98 Escherichia coli SWISS‐2DPAGE database update

Abstract

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

“…Previous observations showed that these mutants were also more resistant (although not always statistically significant) to heat inactivation at 58 to 60°C (16), and this agrees well with the increased heat shock protein expression observed in this study. Comparison of protein expression patterns also revealed differences in proteins other than these heat shock proteins, but we were unable to identify the majority of these protein spots because they were not assigned in the E. coli gene-protein database (19,50,52,55). Interestingly, some studies observed a correlation between heat and pressure resistance among natural isolates of E. coli (1,4).…”

Localizing proteins in the cell from their phylogenetic profiles

“…24) reveal a wide range (700-1,300) of proteins, 13-36% of the proteins expected from the genome sequence of yeast (9). Likewise, whole cell 2D gel analysis of Escherichia coli (25) reveals Ϸ1,846 proteins, 43% of the 4,289 proteins in E. coli (26). Therefore, the percentage of proteins revealed on a 2D gel is Ϸ13-43%, giving 300 divided by 0.13 to 0.43, or about 750-2,300 proteins expected in mitochondria, of the same order of magnitude as we find in this analysis.…”

TheEscherichia coliProteome: Past, Present, and Future Prospects

“…The later use of an immobilized pH gradient (IPG) gel instead of the carrier ampholyte method allowed researchers to apply 2-DE for easier and more-reproducible proteome analyses (25,83). The current use of commercially available 18-cm IPG strips (pH, 3 to 10) along with high-sensitivity staining is generally able to resolve up to 1,000 to 1,500 protein spots in the case of the E. coli proteome (286). However, a large number of the protein spots are found in a 2-D gel of the E. coli proteome cluster at an isoelectric point of 4 to 7 and a molecular weight (MW) of 10 to 100 (294), representing a limitation of 2-D gel separation of unfractionated samples on IPG strips.…”

“…The E. coli 3.5-10 SWISS-2DPAGE map shows 40% of the E. coli proteome (286), among which 231 proteins have been identified by techniques such as gel comparison, microsequencing, N-terminal sequencing, and amino acid composition analysis (Table 2). In contrast, the use of narrowrange pH gradients (pH 4 to 5, 4.5 to 5.5, 5 to 6, 5.5 to 6.7, 6 to 9, and 6 to 11) was shown to potentially display proteins existing at low levels (up to a few protein molecules per cell), resulting in the discrimination of Ͼ70% of the entire E. coli proteome (Table 2; reference 287).…”