Hello,
I want to express two very similar proteins in E. coli. Furthermore, one of the proteins should be produced about 20 times more than the other. For later, I would like to be able to produce each protein separately. Therefore, I was thinking about using two vectors, with one of them harboring a tunable promotor like the pTet promotor.
Does someone have experience in the expression of two proteins in a specific ratio and some tips for me?
Thank you in advance and best regards
Hendrik
Whatever you do at the end there will be some trial and error involved, as there are too many factors out of your control (protein stability, for one, mRNA stability, for another, etc). Also, take into account that in most systems, there is considerable cell-to-cell stochastic variation, so an apparent 20:1 expression ratio when you analyze lots of cells may actually involve significant heterogeneity at the individual cell level.
It seems to me that your best bet would indeed be to make two expression vectors, and in one of them make several different constructs varying the strength of the RBS by using something like the RBS calculator (https://www.denovodna.com/software/), analyzing the resulting combinations to see which one provides the desired co-expression ratio.
Another possibility would be to use a riboswitch. There is the one used in the Ribotite system (Article Dual transcriptional- Translational cascade permits cellular...
), which apparently has the added advantage of minimizing cell to cell heterogeneity and exhibiting dose-responsive behavior (you could put the riboswitch in one construct and vary inducer concentration to achieve the desired ratio between both proteins).Hope it helps!
Simplest one would be to use high copy and lo copy number in a host.
Hi Hendrick, you can also look at that paper by the Busby lab: https://doi.org/10.1093/nar/28.9.1864. They have measured quite different activities in a collection of mutants of a promoter so you might find what you are looking for in that paper. Then the copy number of your plasmid(s) will have a big influence too so I wonder if I would not put the two genes on one plasmid once I found suitable promoters.
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