A possible reason for this behavior is that the protein is in the form of soluble aggregates, which obscures the His-Tag. This may occur because the protein is overexpressed faster than it can fold up. Changing the expression conditions to slow down the rate of expression may help. One way to do this is to express the protein at a lower temperature, such as 16oC, rather than 37oC. Co-expression with chaperone proteins may also help.

Adam B Shapiro

Thanks for your answer

But I am expressing at 20°C. Can 16°C be effective or not?

In my opinion there are 3 optioon possibilities:

1) The his tag in non exposed well: You can try to change the position to the His tag (from C-term to N-term of vice versa) or add a linker beetween the tag and the protein

2) The protein is aggregate and the his tag is buried into the aggregate.

You need to optimize expression conditions (e.g low temperature) or optimize the construct

3) The his tag si degraded and not present in your protein. You can check if by performing a WB using an anti-his tag antibody in your soluble fraction before purification:

4) The overexpressed band that you see is not correspond to your band but to some other higly expressed e.coli proteins. Do you run in parallel a non ijnduced sample or a sample trasfromed with empty vector and induced in the same condition? If not do it or check the idendity of the band from SDS-page using mass-spec.

I'm do not know which of this 4 possibilities adapt to your specific case. To provide an opinion we need to see more data (e.g the pictures of the SDS page acquired in the expression and purification trials)

good luck

Manuele

I don't think switching from 20 to 16 degrees will make much difference.

Do you have any reducing agents, such as DTT, in your sample when you load it onto the column?

Manuele Martinelli

Thanks for your reply

I have checked all these conditions but the problem still exists.

Masoud Kalantar

No, there is no reducing agents