Hi,
I am traying to optimize the expression level of my protein cloned in pBAD vector. Currently, I am inducing it at 0.002 w/v but see a significant overexpression of my protein. The current ribosome binding site in the vector is AGGAGAtatacataccc followed by first start codon ATG.
Can someone suggest will changing the spacer length, currently 11 amino acid (tatacataccc) will change the level of expression? or should I mutate the residues AGGAGA? I want to reduce the overexpression of my protein.
This might be the first time I’ve ever seen a question wanting to reduce protein expression!
1) you could try adding glucose along with arabinose to see if that reduces expression sufficiently.
2) have you checked to see what level expression you get with no arabinose at all?
3) yes by mucking about with the spacer length or the RBS of site itself you should be able to reduce expression. By changing the RBS away from consensus you will reduce its efficiency, or changing the length. I think it is really hard to predict actually what will work so you might want to try a few different constructs concurrently to see what is best.
David Farringdon Spencer
thoughtful research on this deserves a more thorough response. The pBAD vectors were designed to address two issues with lac repressor based vectors. The first is basal expression. Most vectors using lac repressor controls systems are not that tightly regulated and are a bit leaky for basal expression. The pBAD system, because its mode of regulation is based on transcriptional activation instead of repression, is more readily regulated and has tighter control over basal expression. So these vectors tend to be useful when you need to have very low levels of basal expression (essentially off).Secondly the hope was that they would be more tunable and responsive to intermediate levels of inducer (arabinose) so that you can set up situations with partial induction at reduced arabinose concentrations. That also is true and useful but at the population level. In other words if you add very low levels of arabinose you can observe partial induction. HOWEVER, and this is a big thing, it turns out that rather than all cells in the population being slightly induced what is happening is that cells are either ON or OFF and you are shifting the percentage of induced cells in the population. For some things this is fine, but not for others. There is good paper by Siegele and Hu showing this.
The explanation is simple, as a cell becomes partially induced then it's production of the transport protein is increased as well and with more transport protein the cell now more efficiently imports arabinose. So slight induction (randomly) in a few cells results in those cells becoming fully induced. Therefore pBAD vectors turned out not to be great for partial induction schemes.
However adding glucose will always decrease expression through cAMP regulation. This is also true for many lac based systems, but not all because those that use the lacUV5 mutation in the promoter have been rendered independent of catabolite repression (and this is most of them, however not pET vectors if memory serves).
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