Protein complexes perform essential functions in cells. Fatty acid synthase in human is a protein complex made of multiple proteins. RuBisCO is another protein complex. They are composed of multiple proteins. What ideas do you have to express those protein complexes in bacteria or yeasts? Or there might be some other solutions like growing tissue cultures containing the complex proteins of interest?
We tried (unsuccessfully) to co-express an in vivo complex in E. coli using the pETDuet system from Novagen.
Hi there,
Cloning genes as an operon in pET plasmid is a solution to be considered (only one gene leading to tagged protein expression for purification purpose). The obvious thing to do is to perform gene synthesis in order to optimize gene sequence for expression in bacteria.
An other option in yeast is the use of pESC plamid serie (4 plasmids available with 4 different auxotrophy markers) in which you can clone two genes per plasmid under inducible GAL promoter.
To express complex active protein, i suggest to use yeast system rather than e.coli system. e.coli system more simple than yeast system but sometimes e.coli makes foreign protein become inactive after expressed. But must be checked for the protein activity. thanks
My absolute favorite is Agrobacterium mediated transient expression in plants such as Nicotiana tabacum and Nicotiana benthamiana. The disarmed T-DNA vectors can deliver up to 20 kb of recombinant DNA and you can easily put 4 expression cassettes in there. Then you can also very easily coexpress several genes by mixing bacteria carrying different T-DNA plasmids, which allows you to quickly "mix and match" your components of the complex. Finally, its a eukaryotic host and you can express your complex in different subcellular compartments. The procedure is very simple and only requires you to clone the genes, grow the plants and infiltrate/inject them into the leaves.
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