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

4. Translocation

Translocation of the target protein may be advantageous if:
  • S-S bonds are required

  • The protein is hard to purify. There are less than 150 different proteins in the E. coli periplasm and not all of them are expressed. This does usually greatly facilitate purification. There are also systems in Gram positive bacteria that allow secretion into the medium. This sounds ideal for toxic proteins. Many Gram positive bacteria secrete a large number of aggressive proteases into the growth medium (use protease minus strains). The yield of protein is not very high - expect around 1 mg/l culture.

  • Overproduction of membrane proteins is limited by space restrictions of the membrane. However, up to 10 mg of pure membrane protein per liter culture can be achieved.

  • Do not overload the translocation machinery. The Sec machinery itself is weakly cold sensitive.

  • Secretion to the periplasm Not all signal sequences of eukaryotic origin will work in E. colimay be because they often do not follow the positive inside rule There are differences in the efficiency of signal sequences from E. coli If the heterologous protein has a tendency to fold rapidly it may do so before it is translocated. If this is the case try using the TAT system (twin Arg signal sequence) which can secrete folded proteins. In general there is a choice of several (4-5) secretion systems.

    Be aware of the fact that the cell envelope is full of aggressive proteases. There are many protease mutants available that are often helpful (degP and ompT being often useful).