Low expression levels or no expression at all can also be caused by toxicity of the target protein. Protein toxicity can manifest itself on different levels:
Incomplete repression of protein expression. Many promoters are not very tightly regulated and show some degree of expression before the addition of the inducer. Especially lac promoters are leaky. Often this leakiness leads to plasmid instability and/or loss of plasmid (which can be tested using a plasmid stability test). As a consequence the culture will be overgrow by cells that have lost the plasmid (especially when ampicillin is used as a selectable marker) or will not grow at all.
Different approaches can be used to give a more tightly regulated expression:
- constitutive expression of a repressor protein. In the case of the lac promoter by the expression of the lac repressor from the lacI or lacIq gene. For medium copy number plasmids it is sufficient to use a host strain carrying thre lacIq allele. Higher copy number plasmids should contain the repressor gene on the vector.
- use a more tightly regulated promoter, e.g. the arabinose promoter ( PBAD).
- use a lower copy number plasmid.
- constitutive expression of phage T7 lysosyme from a compatible pLysS or pLysE plasmid (Novagen). Lysosyme binds to T7 RNA polymerase and inactivates the enzyme. After the addition of IPTG the expression level of the polymerase will be much higher than that of lysosyme and this will overcome the repression.
- addition of 1% glucose to the culture medium to repress induction of the lac promoter by lactose, which is present in most rich media (such as LB, 2xYT).
Besides tightening regulation (see above), other approaches exist to prevent losing the expression vector from the cells.
- use of elevated levels of antibiotics (up to 200 mg/ml
- use the "plating" method for inoculating cultures. Cultures are inoculated by scraping off agar plates (see protocol).
Toxicity upon induction. Some proteins are so toxic for the cells that they do not only inhibit growth but also kill them. Thus, almost immediately after induction cells die and expression levels will be very low. Several approaches are possible to decrease the effects of protein toxicity:
- periplasmic expression. Secretion of the target protein to the periplasm (or the medium) allows for the accumulation of proteins that are toxic in the cytoplasm.
- expression in inclusion bodies. Although difficult to direct it is advantageous to express toxic protein in inclusion bodies. In aggregates the proteins are not toxic for the cell and they can be obtained by in vitro denaturation and refolding.
The expression of hydrophobic heterologous proteins (usually membrane proteins) is often toxic to the bacterial host. The following approaches can be used to lower its toxicity:
Express only the hydrophilic domains.
Use special host strains. Some host strains, e.g. C41 (DE3) and C43 (DE3), deal better with membrane proteins than it parent BL21 (DE3).
Reference: Miroux, B. & Walker, J.E. (1996) J. Mol. Biol. 260, 289-298.