His tagged protein expression

Antonin Tutter atutter at aim.salk.edu
Sat Aug 8 02:59:16 EST 1998


>I am trying to express a 15 kDa protozoan protein as a His tag fusion in
>Qiagen's pQE30 vector. My problem is that the protein is being expressed
>as a trimer (possibly!?) as the over expressed protein appears to be about
>45 kDa on the SDS-PAGE. This high molecular weight protein gets purified
>on the Ni-NTA resin supplied by Qiagen. 
>Can any one offer ideas so as to why it may be happening and what can I do
>to get the protein of the right size? I have confirmed the construct by
>sequencing the whole of the insert and the junctions.

The problem may be caused by Ni++ leeching into the elution buffer, and
acting as a multimerization point by binding multiple peptides. Try adding
EDTA to your eluted protein and see if it continues to happen.




_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  atutter at aim.salk.edu
web:  http://www-biology.ucsd.edu/~atutter/




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