Hi All, I am working with a methyltransferse protein and its later determined crystal structure indicates its substrate SAM was bound during protein expression or purification since no SAM was added during protein crystallization. Now I want to obtain the apo form protein for further characterization. Here are the strategies I can think of:
1. Mutate residues that are possibly critical for SAM binding. The shortcoming of this strategy is the protein is modified, also mutating one residue may or may not be able to get rid of SAM binding. 2. partially denature protein with denaturing reagent such as guanidine. This strategy is hoping the partial denaturing would loose SAM binding, then dialysis out SAM, reconcentrate protein and re-run sizing column to get rid of aggregated protein. This strategy may only work with well-behaved protein. The harsh denaturing reagent treatment could lead to total protein denaturing. I am asking more experienced biochemists if there are other better strategis for obtaining apo protein that I am not aware of. Thanks very much for sharing your knowledge. Eric
