Hi Angela,

Yes, fusion proteins can be isolated from eukaryotic cell cultures, but antibody-based columns are both more expensive and more difficult to optimize than Ni/Co-based resins used for 6xhis tags (6xhis fusion proteins are normally denatured during column purification which exposes the tag). Also, if you are using human cells, the Myc antibody will also co-purify the Myc protein. The effect of the addition of tags (both small like Myc and HA, and large like the transactivation and DNA-binding domains of your Y2H system) always have the potential for having deleterious effects and may have to be determined empirically in each case. This is true for both eukaryotic and prokaryotic synthesized proteins.

The main advantages of eukaryotic expression is potential for essential post-translational processing and modifications, and likelihood of correct folding. Advantages for prokaryotic expression is yield (cost and scalability), and lack of contaminating eukaryotic proteins.

Can you do the experiments with whole cell lysates without purification?

In the big picture, recloning to enable prokaryotic expression to generate significant amounts (mgs) of recombinant protein may be a relatively minor part of the effort for the entire project.

For me, it would depend under which promoter you have cloned your protein for the expression. Unless you have it under a strong, inducible promoter (eg GAL1,10), I don't think you will get a lot of protein for purification. It will be enough for yeast two-hybrid of course, but it would complicate further use of the protein for purification and downstream use.