Isn't it that the expression depends on the promoters, the specific functions and so many other factors (e.g. whether the proteins are constitutively expressed ones, organelles specific, pathways specific, cell cycle specific etc.). If they are constitutively expressed genes at all the prot-transcriptional levels, then I don't see any reason why they are not simultaneously expressed. However, if one of them is normally suppressed based on the cellular conditions, then you would see a difference in expression levels. Don't know whether this answered your question.

If you use the baculovirus-insect cell system, the chances are very high.

Just M. Vlak

Dept. Virology

Wageningen University

Dual protein labeling is a common approach, where m-cherry and GFP (most common fluorescent proteins) or any other fluorescent proteins are expressed simultaneously or condition based manner.

If you absolutely have to have both expressed, you can use one promoter and use an IRES.

We routinely express two or more proteins in Sf9 cells in order to generate e.g. active CDK-cyclin complexes and frequently get good results, so I can only assent to Just's comment about using the bacuovirus expression system. If you would like to express your proteins transiently in mammalian cells you should consider to use an IRES construct to avoid problems with using two separate plasmids/promotors. In general the result will always be highly dependent on the individual protein.

I have never expressed proteins in cell lines, but expression of two proteins from bacterial cultures is routine. You can have the DNA encoding for the two proteins on separate plasmids with different antibiotic resistant markers. After transformation of the cells with the two plasmids and when growing the cells, you must include both the antibiotics in the culture medium so that only the cells transformed with both plasmids will be selected. Alternatively, if it is better suited, you can have the two proteins on the same plasmid with the same kind of inducible promoter. You could use similar strategies in cell culture. Unless the proteins form a complex with each other that is toxic to the cell, there is no reason why this cannot be done.

Good luck!

Daniel, Thanks for your answer. When you mentioned CDK-Cyclin complexes, how are they complexed? When they are produced in same cell line, are the produced in complex or are there other manipulations required. Are these complexes covalently complexed or some other interaction.

Thank you for your help

I'll echo what Czuee wrote. We do this in E.coli all the time using two plasmids with different antibiotic resistances, or with both proteins on the one plasmid. In the first case (two plasmids), we're expressing calmodulin and one of its binding partners. These form a non-covalent complex in the bacteria. In fact this complex is strong enough that we can purify it without it falling apart. In the case of the two proteins on one plasmid this forms a heterodimeric enzyme.

Jeffrey, the CDK-cyclin complexes are "real" complexes i.e. the subunits stick together by non-covalent interactions, as we could show by affnity-purifying them under native conditions with the affinity tag only present on one of the subunits and the other subunits being co-purified. They are also exhibiting kinase activity which in itself shows that the cyclins are functionally complexed to the CDKs they activate. It works in our hands for most of the known CDK-cyclin complexes, even for triple complexes like CDK7-cyclinH-MAT1.

This is commonly done in bacterial system. In principle it should also work with mammalian or other cell line. Please check the link below to know, how it has been done in bacteria.

http://www.sciencedirect.com/science/article/pii/S1046592810003037

Daniel, do you use co-infection to generate your complexes, or a multi-bac system?

Dear Jeffrey,

During my thesis (close to 2k), we expressed 2 proteins with one promoter and IRES in-between, inside CHO cells. We also added some gylcose-related enzymes on a different plasmid with lower succes and way lower reliability... We used CHO cells, infected with one plasmid and, when stabilized, infected with a second, based on the experiments we wanted to do.

So, Daniel Mueller advices should work!

Dear Scott,

actually we routinely use co-infection - it works well in our hands and one has the advantage of being flexible with the combination of different viruses.