soluble protein expression in E. coli

Koen De Smet kds30
Wed Jan 17 07:30:47 EST 1996


john brennand
<john.brennand at gbapr.pharmaceuticals.zeneca.tmailuk.sprint.com
> wrote:
>Saverio
>
>yes we have had the same problem with a number of proteins.  We (or
>rather our E Coli expression & Protein Science groups) solved our
>problems by applying a  "Low-Slow-Long" protocol.
>
>Basically, they lower the growth temperature to 22o - they induce with
>iptg for longer (overnight) and lower the iptg concentration (~50uM).
>Playing around with these conditions allows soluble protein to be
>accumulated - it is usually at much lower levels though (0.1 - 1% of
>total protein) so purification is essential (6xHisTag for us)
>
>Try it
>
>John
>
>
We tried it, but it didn't work for us. We had a mycobacterial  ORF expressed in pQE30 (Qiagen) producing a 6xHis tagged protein. At 0.035 mM we still had as much expression as at the standard 1 mM and it remained insoluble. Other conditions did not result in expression of soluble protein either (room temperature, adding MgCl2,...).

We harvested the insoluble protein and denatured it with 6 M urea. This was then purified on a Ni++ column in hte presence of urea. The urea was then removed by stepwise dialysis against buffers with 4M, 2M, 1M, 0.5M and no urea. The protein was soluble and had some enzyme activity (but not as much as we would have expected).

Maybe this approach helps?

Koen De Smet





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