Hey guys! I'm working on a huge complex, trying to purify it for structural studies. All of the components need to be cloned into one vector, and there are over 20 of different proteins in it. Does any of you have any experience with cloning such large complexes, but in a way that introduction of new tags is easy and straightforward? What I mean is, I want to try to purify the entire complex, but I don't know which protein will be the base protein that would catch all of the others, so I'd probably have to introduce a tag to each of them separately and see which one works best. I'm looking for something like an easy subcloning system that would work with such big constructs.
I have no experience in expressing such a big complex from recombinant proteins, but I am a bit skeptical about the possibility of successfully expressing them all in a single clone and forming the complex. If it doesn't work, you will have to clone the genes and express the proteins individually to reconstitute the complex in vitro.
This is one of those situations where a lot of advance planning for experimental design is a good idea. Are these 20+ proteins all in an operon so that you can clone them as one? Or is this not even bacterial?
You might consider building an expression vector that expresses all 20 and then a second plasmid with each protein one at a time with so you can determine which is the best one to tag. But getting all 20 expressed is going to be a challenge.
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