Any idea of the affinity of the complex? If weak enough, you could
just dialyse, dialyse, dialyse... Or, you could bind your protein to a
column, and wash with a slow-flow of appropriate buffer. Or you could
also try to compete it out with adenosine, for example, which
presumably has a lower affinity, but at high concentrations would
still compete it out, and then dialyse or rinse while bound to a
column.
Jacob
On Fri, May 20, 2011 at 10:18 PM, jlliu liu <[log in to unmask]> wrote:
> 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
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: [log in to unmask]
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