When studying a certain protein, purifying the protein fused with His tag can obtain a high concentration of protein, but precipitation is prone to occur. We found that whether this protein is fused with GST or pET32a at the N-terminus or mcherryh or GFP at the C-terminus, it can be expressed normally, and it will not precipitate at a high concentration.

The reviewer asked to know the oligomerization state of the protein. I initially used the His tag protein directly and found that it was highly polymerized (maybe greater than 30), and the UV280 response value was very low. We guessed it should be in the SEC column The protein aggregates. Later, we used the mcherry fusion tag to conduct experiments and found that the protein fusion mcherry was in the form of a tetramer, while the individual mcherry protein was in a monomeric state. Is this method reliable? For this type of protein, do you have other better methods to detect the oligomerization state of the protein?

Thanks a lot for your answers and help.