We are interested in coexpressing pairs of proteins in E.coli. We have them on a couple of typical commercial plasmids but there is an issue of ori incompatibility or the antibiotic resistance. Before continuing to bang our heads against the wall, I am sure there will be experts in our ResGate community with suggestions
Thanks
Gonzalo
Gonzalo de Prat-Gay, following Xi Jiang's suggestion, I also recommend you reading this work by Hagan, Article Reconstitution of Outer Membrane Protein Assembly from Purif...
, in which they used multiple plasmids (including a pETDuet) to express the different components of a five-protein BAM complex. Good luck for your project!
The suggestions above are certainly fine and Duet works well. But you also don't need to get complicated. You can just put the two genes adject to each other and drive from a single promoter. If you include the RBS, ATG, ORF and stop codon from the second and put it downstream of the first it should be fine. Remember that bacteria use operons all the time. Just don't have too much spacer in between the two genes.
Thanks Michael!! we are doing something similar to what you say, we will get to the expression stage some time soon. Do you have a tested sequence of the exact composition and length (ie between stop and ATG) that work for any pair? Or which is the length (range?) you recommend?
you need to be sure and bring the RBS sequence along with the ATG to get good translational initiation for the second gene. If it is cloned in an expression vector already you can just pull out the entire cassette. This gives you two independent translation units from one mRNA. I think 20-100nts is fine as spacer between end of first ORF and start of second.
I have used PET-Duet1 for co-expression of recombinant protesses and artifical GFP based substrates in competent E.coli . You may check out this online article we published recently (www.mdpi.com/2076-0817/10/2/102/pdf). Good luck.
A possible alternative to the pDuet are the pQlink vectors, designed from Konrad Bussow.
https://pubmed.ncbi.nlm.nih.gov/17311810/
that are available from ADDGENE
https://www.addgene.org/search/catalog/plasmids/?q=pQLink
i had the opportunity to test it in 2009 when i visited for a months the laboratory of professor Udo Heinemann in Berlin and it works very well.
Good luck
Manuele
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