I use Pichia pastoris for recombinant Lipase enzyme from Candida sp. production (secreted into medium).
I try to follow the graduated members that do this project, unfortunately the protocol not give much detail, and the result cannot repeat (in my case).
I face the problem in the step of 1. little dissolve of protein pellets in buffer, and 2. enzyme activity.
First, after get the cultivated medium contains lipase enzyme, I add the ice-cold ethanol to 80% final concentration and stiring at 4 Deg C for overnight. Then, get a lot of white precipitated pellets after centrifuge. (This step is fine.)
Second, I try to resuspend the protein pellet in 100 mM Potassium phosphate buffer pH 7.2, but It can dissolve very little amount. Normally, the protein precipitated in buffer.
Finally, I use the solution (only supernate) for assay the enzyme activity. It show very little activity.
The question are
1. I guess ethanol will denaturing enzyme, so the enzyme will precipitate in denatured form? and non-function?
2. If enzyme was denatured from ethanol. Which method use for renature of enzyme?
3. I see someone use 8M urea as buffer, and add 9 volume of ethanol. This method is it works for renature enzyme?
4. Should I change to use ammonium sulfate precipitation method? for functional enzyme
P.S. the protein don't have any tag, and I would like to use common and easy method for protein (not HPLC, or column)
Any suggestion, please suggest.
Thank you,
Tatpong
I would use ammonium sulphate instead of the ethanol, it is safer for activity of the protein.
Hi
Undissolved precipitates are denatured protein, stirring with high speed could cause the same thing with ammonium sulphate so use gentle stirring usually over night at 4C.
Good luck
You are correct about item #1 in your summary. Item #2 is unlikely to happen but you could try to solubilize the precipitate with urea at a low enough concentration to just get it to dissolve then try to refold by dialyzing out the urea. The method will depend upon how rugged the enzyme is and a lipase may not be very rugged and could be irreversibly denatured. The urea will also denature the protein but you are firstly trying to solubilize the precipitate and then hoping that removal of the urea will allow the enzyme to fold back to its native conformation. That's asking a lot for an enzyme. the refold should actually be done as a partial factorial design by changing other components ad their concentrations such as salts, pH, co-solvents, temperature, lipid additions, etc In Item #3 the ethanol seems to be denaturing knowing that lipases are water soluble and act only at the interface of emulsified lipid droplets. The 8 M urea will denature the enzyme but then the urea is diluted to less than 1 M urea by ethanol (assume very cold) to possibly allow the precipitation of product but keep other contaminants in the urea/ethanol phase. It may be possible that the product is in the ethanol/urea phase and contaminants are precipitated? You are still faced with the issue of removing the ethanol and urea for refolding or dissolving the product in the case of precipitation. In item #4 you may have no other choice than to use column chromatography if you want to have enzyme activity. If your process is for industrial application and you're trying to cut costs then keep in mind that column chromatography, even single use or continuous processes, may actually be cheaper and safer and have greater product purity due to regeneration cycles or the elimination of cleaning validation or safety issues with organic solvents.
Dear Soskine, Albaser, and Shimamoto,
Now, I try to use Ammonium Sulfate precipitation insted of Ethanol precipitation.
I do only a small scale (50 or 100 mL of medium culture). If I do much for industrial scale, I will use colum chromatography like you suggest.
Thank you for your suggestion.
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