Actually i am trying to express and purify a microbial metalloprotease. I successfully developed the construct and cloned it in E.coli cells and the protein got expressed in form of inclusion bodies.
Purified inclusion bodies dissolved in 8M Urea containing buffer and then refolded by step wise dilution in refolding buffer.
when i plated inclusion bodies dissolved in 8M urea and refolded IB in 1% casein agar plate. Both of the cups are shpowing activity?
I thought may be urea is denaturing casein and checked the only 8M urea containing buffer in Casein agar plate but it is not showing activity.
How is it possible for a protein to show activity in presence of 8M Urea?
Am i doing something wrong? suggestions are welcome.
Once the content of the cup with urea + enzyme diffuses into the neighbouring casein agar, the urea concentration will drop, apparently to an extent that is sufficient to renature the enzyme
Thanks a lot sir, i were the same thought after this, but i needed an expert opinion to be confirm.
I have the same experience when I tried to purify peptidyl-prolyl cis-trans isomerase. Theoretically, 8M urea denatures the protein structure that may not the determinant of enzyme function. In contrast, sometimes amino acid sequences are the key of enzyme activity even in linear form. Please check the reference I linked.
Good Luck~
http://www.sciencedirect.com/science/article/pii/S1046592814000849
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