Using size exclusion chromatography I have shown that two proteins from one pathway form a complex. How can I co-crystallize these two proteins together? Shall I purify them separately and then mix them together or they should be co-expressed together?
You can try both. Some only form a complex during synthesis like HSV gH/gL. But if they form a complex after each is purified separately that would be interesting. But for co-crystallization, they should run together by size exclusion and then you can pool the large MW fractions, concentrate and run crystallization trials. If they do not have high enough affinity to stay together by gel filtration, they will likely not stay together as a co-crystal. You could also look by EM (simple negative stain or hook Fabs to each and look.
You can try both. Some only form a complex during synthesis like HSV gH/gL. But if they form a complex after each is purified separately that would be interesting. But for co-crystallization, they should run together by size exclusion and then you can pool the large MW fractions, concentrate and run crystallization trials. If they do not have high enough affinity to stay together by gel filtration, they will likely not stay together as a co-crystal. You could also look by EM (simple negative stain or hook Fabs to each and look.
If you can purify the complex, you should coexpress them together. That should be the most stratforward way if you have no other concern.
My experience was purifying the protein separately, demonstrating their interaction by bichemistry study such as activation of enzymatic activity, and mixing them together for crystallization screening. They might not form stable complex on SEC, but they might come together at certein crystallization condition and give you crystal if you are lucky. I have cryslaized complex with three proteins in this way with excess molar ratio of the smallest protein. Good luck!
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