I constructed a pET-53 to express yeast gene in E. coli BL21(DE3). The culture condition was culture the BL21(DE3) until OD600 = 0.4
If the protein is in inclusion bodies, it will almost certainly be necessary to denature them in 8M urea or 6M guanidine HCl, then refold the protein to regain activity. While working on this, you may want also to try other ways of expressing the protein to try to find one that gives soluble protein.
I constructed a pET-53 to express yeast gene in E. coli BL21(DE3). The culture condition was culture the BL21(DE3) until OD600 = 0.4 - 0.8 then add IPTG and culture overnight at 18 C. I found that my protein located in the inclusion-body, so, how can i extract and purify this protein without losing any enzyme activity? Thank you.
If the protein is in inclusion bodies, it will almost certainly be necessary to denature them in 8M urea or 6M guanidine HCl, then refold the protein to regain activity. While working on this, you may want also to try other ways of expressing the protein to try to find one that gives soluble protein.
I think you need to change your culture condition to gives soluble protein, for example decrease your concentration of IPTG and the culture degree. It is very difficult to get the activity protein in inclusion bodies.
If your over-expressed protein is going into inclusion bodies, then one approach you could take is to isolate your protein from the inclusion body, denature it in buffer containing 6M guanidine HCl and then refold it slowly to regain proper fold and activity. This is a tricky technique and many would rather work around changing their culture conditions to somehow obtain soluble protein. But life isn't always easy.
Instead of refolding, you could also try increasing the solubility of your enzyme by fusing it to a robustly soluble protein, such as MBP or GST. However, even if the fusion is more soluble than your enzyme alone, this does not guarantee that the enzyme is correctly folded, so you will have to check for activity.
Do you need to express the protein in E. coli? Have you considered expressing it in yeast as this is the natural host?
Thank you everyone. How's about co-transfrom these two subunit together in the same cell and expecting that they will reconsitution themselves and I can measure the enzym activity from the inclusion-bodies. Notably that I have tried LB+1% glucose and M9 with several IPTG concentration but it still form an inclusion-bodies.
Some people seem to think that enzyme activity can be present in inclusion bodies. Here are 2 paper on the subject:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1224866/
http://www.ncbi.nlm.nih.gov/pubmed/18388459
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