I would try the following:
1. Remove the His tag.
2. If you need to get rid of the imidazole I would try adding ammonium
sulfate.
3. Is your protein proline- and/or rich in aromatic residues? This may
explain your need for imidazole and solubility.
> Hi there,
>
> Sorry for the off topic questions. We need your feedback.
>
> We are expressing a rat protein in insect cells. It is expressed as a
> secreted protein with an N-terminal 6xHis tag. We can get about 4 mg of it
> from 1L culture and everything looked quite normal at the very beginning
> (at 4C). When I changed the buffer to HBS using centricon to get rid of
> imidazole (@ 4C), I noticed that it took a long time to concentrate and I
> saw some ppt. However, when I took some of it (at about 1.2 mg/ml) and
> kept them at room temperature, the solution turned cloudy in a few
> minutes. I tried to change the pH by diluted in 1M stock of different
> buffers (pH 4.5-8.5), change the NaCl concentration, and add 10% glycerol,
> but it still crashed out at RT. However, it seems OK, I hope, when
> kept on ice. I am wondering whether any of you had a similar experience
> before. It is not a problem for us to do everything at 4 degree. I just
> worry that it may indicate something wrong with this protein. The protein
> should be stable since it has
> been shaking at 27C for four days…
>
> Many thanks.
>
> Best,
> Chen
>
>
>
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