I am working on a protein fragment and eventually I am going to crystallize it with another protein. I read one paper about the crystallization of that fragment and it said the carboxymethylation is essential for crystallization. Any idea, advice, or protocol would be appreciated. Thanks in advance
What residues are being modified? Carboxymethylation is often used to block cysteines, important for example in a cysteine protease to kill the enzyme and stop autoproteolysis. What rationale did your paper give for the modification, is the carboxymethyl group visible in the structure? There are readily available protocols online and in the literature.
First, Thank you so much for info response. I have not modified anything in my protein. Basically, I would like to crystallize my protein fragment and other interesting protein ( both at the same time to proof the binding between them). I do not know if the cys.group is visible or not. Is there a program or tool to investigate that? If you have the carboxymethylated protocol under your hand, please send me a copy. Regards.
Ahmed
I work with the ligand binding domain of the estrogen receptor alpha. Researchers used to carboxymethylate the protein prior to crystallization because there are solvent exposed cysteines which tend to cause misfolding. We've since found that adding a reducing agent like 2-mercaptoethanol (I keep 15 mM in my buffer) is preferable to carboxymethylating. Hope this helps!
Thanks Mr.Fanning. Do you crystallize only E domain ? If yes, how much protein did you use to do that? Regards
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