Purifying protein with no his tag - (Mar/16/2008 )

Hi,

I have expressed my protein in E.coli after subcloning it to remove His-tag. I need to crystallize this protein which is nearly 15 KDa size. However, I am not able to get the pure protein after DEAE column purification and I am getting a extra peak of possibly nucleic acids at 228 nm and my protein and the impurity elute at the same time in 100 mM NaCl. I tried gradient salt elution an also pH adjustments but it did not work. Can anyone help me with this.

Thanks
kush

If you have nucleic acid contamination, try treating the crude lysate with DNaseI at the start of the process. What makes you think it's DNA? OD260?
You could run the de-His tagged protein(s) through a gel filtration column, rather than DEAE.
You could try using a strong ion exchanger, such as MonoQ or MonoS.
If the protein (if that's what it is) elutes at the same pH and salt, could it be a dimer?

Thanks for your reply.
I will ty the DNase treatment for the crude lysate. Gel filtration and MonoQ column are expensive.

Thanks

Its less likely to be DNA. It binds much more tightly and requires higher salt conc to elute off. Rarely one can get a protein in its pure form with a single column step without the use of an affinity purification principle.