GST-fusion protein is insoluble.

Alexander N. Kukushkin kan at mmcd.cyt.ras.spb.ru
Tue Dec 17 11:32:23 EST 1996


>   Dear Netters:
>     I am trying to express a gene from rice in E. coli, using the GST-fusion
>   protein system, but perhaps due to its high Cys content (17 Cys out of a
>   total of 131 a.a.), the expressed product is insoluble. How should I
>   solublize and renature it without the loss of its affinity to the
>   glutathione-agarose?
>     Thanks for any help in advance.
>
Try the following procedure:
1. Gather E.coli cells by centrifugation;
2. Suspend the cells in PRS;
3. Add DTT to 5 mM (for example, out of 1M stock);
4. Add lauril-sarcosyl to 2-3% final concentration (10% stock);
5. Sonicate;
6. Add Triton X-100 to 4% (20% stock);
7. Centrifuge;
8. Take the supernatant and use it for binding reaction with the glutathione-
agarose.
Good luck
Alex




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