I am purifying RNAse E in E. coli (118 kDa). I cloned the rne coding sequence on pET28a with a His tag at the N-terminus. The protein overexpressed fine (see attached figures). The binding buffer contains 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, and a proteinase inhibitor. The elution buffer contains 20 mM Tris-HCl (pH 7.5), 300 mM NaCl, and 250 mM Imidazole. I tried binding at both room temperature and 4 degrees Celsius for 1 hour to overnight, using low and high salt concentrations in the buffer. However, the amount of protein that bound to the beads was very low. I have performed several His-tagged protein purifications before, but I have never experienced this issue.
One possible reason is that the protein is in an aggregated but soluble form. Other possibilities are that the His-tag was lost due to proteolysis, or it is buried in the protein. It may be worth trying a construct with the His tag at the other end of the protein.
You can check the alphaFold prediction with you protein plus tag to see if the tag is accessible, or if the tag would be better positioned at the other terminus or a linker sequence is beneficial. It might be that you have to check other pH (check the ip of the protein and try one below and one above for starters) or other buffer systems as for example phosphate or HEPES. That might improve stability of the protein.
I have found the using HEPES as the buffer helped significantly. Tris can change pH with changes in temperature. Is your protein soluble? You will see your protein in the pellet as inclusion bodies if it is not soluble. Additional things that you can try is perform your purification under mild denaturing conditons (2-3M Urea) to slightly unfold your protein and make the affinity tag more accessible. As previously suggested, cloning your His-tag at the other end of the protein may help as well.
Adam B Shapiro Tim Habenicht Ryan Yellamaty Thank you all for your suggestions! I have reconstructed the sequences with the 10xHis-tag at the C-terminus, along with a flexible linker, Gly-Gly-Gly-Ser. I will also try using HEPES. My protein is soluble but the binding is low. So I think maybe the tag is hidden. I will provide you guys with an update soon!