The effect of expression of foreign proteins on the physiology of the bacterium <I>Escherichia coli </I>has been investigated. To quantitate accurately protein production a model system was developed based upon the expression of an α-2 Interferon fragment, (amino acids 4-155). A polyclonal antibody-based enzyme linked immunosorbant assay (ELISA) for α-2 IFN was developed for rapid determination of accumulated protein. As with many foreign proteins expressed in <I>E. coli,</I> α-2 IFN (4-155) was accumulated in an entirely insoluble form as an inclusion body. For accurate determination of α-2 IFN (4-155) by ELISA, protocols for the solubilisation of the foreign protein were developed. Foreign proteins could be accumulated at up to 30% of total cell protein with no significant difference in the specific growth rate of the recombinant cell compared to its parent. Determination of RNA: protein ratios indicated that the protein synthetic capacity of parental and recombinant cells were not significantly different. A model is proposed in which the expression of certain proteins was reduced to accommodate the extra translational load of recombinant protein production. Since RNA pools were unaffected by recombinant protein production it is inferred that the ribosomes and other proteins involved in translation are not significantly affected. The data predict that many <I>E. coli </I> proteins are synthesised at rates faster than those needed to sustain specific growth rate. The susceptibility of recombinant cells to environmental challenge is, however, increased indicating proteins, that are accumulated to lower levels during foreign protein expression, have a role in the ability of <I>E. coli</I> to adapt to a changing environment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:241448 |
| Date | January 1994 |
| Creators | Hunter, James Neil |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=191898 |
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