Characterization of periplasmic Escherichia coli protein expression at high cell densities

We have used two-dimensional electrophoresis (2-DE) to analyze changes in p rotein expression profiles during a microbial cultivation process on an ind ustrial scale. An Escherichia coli strain W3110 containing the gene for rec ombinant human growth hormone production was used. Samples were taken at ti me intervals ranging from fast to slow growth rate (late growth phase at hi gh cell density/starvation) and 2-DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression lev els of a number of proteins. Twenty-four protein spots were identified usin g a combination of matching with SWISS-2DPAGE E. coli map, N-terminal seque nce analysis and mass spectrometry matrix-assisted laser desorption/ionizat ion (MALDI). Two of the most abundant proteins expressed at late growth pha se (pl 5.4/28 kDa and p/5.5/28 kDa) were subjected to N-terminal sequence a nalysis after electrotransfer of the proteins from a preparative 2-DE gel t o polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino ac ids in combination with approximate p/ and M-r identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots we re subjected to tryptic in-gel digestion and analyzed using MALDI. Peptide mass fingerprints from both spots showed similar MALDI spectra and 10 of 10 tryptic fragments confirmed the identity as deoC. The identification of th e acidic variant of deoC on 2-DE gels and the observation of this variant a s induced during late growth phase is novel.