The Art of Gene Expression in E. coli



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Gene expression in
E. coli




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Tilman Maier og Lars Jessen
1. Factors that influence Gene Expression
2. Expression vectors
3. Folding
4. Translocation
5. Protein stability
6. How to address the hopeless case
7. Safety
8. Links

6. How to address the hopeless case
  • Arrays (transcriptional profiling) on the expression strain with and without the overexpressed recombinant gene will point to chaperones to co-overexpress or protease mutants to use.

  • Phage display to optimise interaction between subunits

  • Genetic selections to generate mutants that functionally overexpress the target gene (e.g. Production of active and soluble Serine Phosphatases can be selected in serB mutants. serB mutants do not grow in minimal medium lacking serine. Active Serine Protein Phosphatases will complement the serB mutation. Reference: Hoffmann et al. 1994. Yeast. 10: 567-578. entrez/medline

  • If expression in the periplasm does not work because the required chaperones are absent try to screen a gene library of a bacterium that has a larger genome such as Pseudomonas or Mesorhizobium loti , a larger number of periplasmic proteins, and thus more likely a more complete set of chaperones and folding catalysts.

  • What if the activity of the produced protein is toxic to cells? Try to isolate chromosomal suppressor mutants, use a very tight promoter, induce late (at high OD)...