5 replies to this topic

[micha]

Hello,
I am a rookie for protein purification and I am doing his-tag protein purification. My protein is around 26.1kDa and PI is 7.1.  After purification, I ran SDS PAGE and found that my protein is in the cell pellet, not in liquid part.  I expressed the protein at room temperature for 4 hour with 1mM IPTG.  Could you let me know how to solve this problem, please?  I have worked for more than 7 months and I am very discouraged.

Thank you so much in advance.

[Linnea]

If you get inclusion bodies there are techniques you can use. I've never done it myself, but if you use the google search engine you find stuff like this: http://www.spineurop...ng_protocol.pdf

If you got to have your protein in solution you could try lysing the cells with 8M urea.

Good luck.

Edited by Linnea, 21 March 2005 - 06:13 AM.

[micha]

Thank you so much!!!

[Linnea]

Observe that 8M urea will denature your protein, so if it is important that it is native you have to do some refolding.

[sharath]

Overexpressed protein appears in the cell pellet when it forms inlcusion bodies instead of soluble form. this is a common problem during overexpression of proteins which require extensive post-translational processing. During overepxresson, the rate of translation overtakes procesing of the nascent polypeptide and consequenlty, a bulk of unfolded proteins accumulate and aggregate as inclusioon bodies.

The first strategy to solve the problem would be to try avoiding the formation of inclusion bodies by slowering the overexpression. Grow the cells at 30C,25C,and 20C. harvest cells at 30mins interval after induction and check for activity in the soluble form.  If these tricks dont yeild try subcloning inot a less powerful expression vector.

If nothing works then go for solubilization of inclsuion bodies. This works well with some proteins but not always. There are many aggregate solubilization solutions available as a kit from Hampton Research.

[leekaming]

Maltose binding protein (MBP) may increase the solubility of proteins. You can try to clone your gene into MBP vector.