His tagged protein expression
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.
Salk Institute for Biological Studies
10010 N. Torrey Pines Rd.
La Jolla, CA 92037
email: atutter at aim.salk.edu
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