Efficient expression of recombinant protein was achieved in E. coli through the use of vectors capable of copy number amplification. Expression was regulated by the tryptophan promoter and plasmid copy number by the temperature sensitive Lambda Pg promoter. Reproducible expression of a variety of recombinant proteins was achieved in defined medium under fermentation conditions. In all cases, cell growth was strongly inhibited after amplification of both plasmid DNA and product expression. Plasmid copy number control was studied in continuous culture. Below 36°G, plasmid DNA was maintained at a low and constant level. At 37°G and 38°C however, plasmid DNA amplification was activated. In order to study the effect of physiological parameters on expression from these plasmids, the CAT protein was chosen as a model. The effect of specific growth rate on copy number amplification was studied in batch and continuous culture. Prior to induction, higher growth rates increased CAT expression. After induction however, clear trends were difficult to determine although growth rate clearly influenced the lag period before DNA amplification occurred. In continuous culture, a combination of reduced growth rate and overgrowth by the plasmid free cells caused transient product formation. Therefore to conduct extended experiments under inducing conditions, two stage continuous culture was evaluated. Cells grown at 34°C were fed continuously to a second fermenter held at 38°C. Stable production was achieved over a 160 hour period - sufficient time to perform reproducible experiments. In these experiments, specific growth rate in the second stage was determined to be a significant factor influencing high level GAT expression. In addition, the efficiency by which it was both transcibed and translated from the plasmid DNA could be altered. The residence time in the second stage played a secondary role - mainly influencing the point at which plasmid-free cells could overgrow the culture. Trends in plasmid stability were also studied. The development of more sensitive methods to detect plasmid instability revealed the gradual accumulation of plasmid-free cells in these cultures. This was due to overgrowth of these plasmid-free cells rather than plasmid loss due to segregational deficiencies. Higher rates of overgrowth were observed at greater product expression levels.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:253995 |
| Date | January 1990 |
| Creators | Brown, Michael Edward |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/843792/ |
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