Characterization of periplasmic Escherichia coli protein expression at high cell densities.
We have used two-dimensional electrophoresis (2-DE) to analyze changes in protein expression profiles during a microbial cultivation process on an industrial scale. An Escherichia coli strain W31 10 containing the gene for recombinant human growth hormone production was used. Samples were taken at time intervals ranging from fast to slow growth rate (late growth phase at high cell density/starvation) and 2-DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression levels of a number of proteins. Twenty-four protein spots were identified using a combination of matching with SWISS-2DPAGE E. coli map, N-terminal sequence analysis and mass spectrometry matrix-assisted laser desorption/ionization (MALDI). Two of the most abundant proteins expressed at late growth phase (pI 5.4/28 kDa and pI 5.5/28 kDa) were subjected to N-terminal sequence analysis after electrotransfer of the proteins from a preparative 2-DE gel to polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino acids in combination with approximate pI and Mr identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots were 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 the acidic variant of deoC on 2-DE gels and the observation of this variant as induced during late growth phase is novel.
Franzén
B
B
Astra Arcus AB, Preclinical R&D, Södertälje, Sweden.
Becker
S
S
Mikkola
R
R
Tidblad
K
K
Tjernberg
A
A
Birnbaum
S
S
D000180:Acrylic Resins; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis*; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D019897:Periplasm / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000180:Acrylic Resins; D001426:Bacterial Proteins; C016680:polyacrylamide gels