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Protein Refolding - Precipitation during dialysis - (May/26/2014 )

Hello All,

 

If anyone has suggestions below, this would be welcome.

I am trying to pioneer a refolding protocol for an insoluble protein in our lab for the first time.

 

 

The problem:

 

I have a protein that expresses as inclusion bodies (not soluble). The pI of the protein is 6.7 and it is His-Tagged.

The protein solubilises well in 8M Urea containing 1mM DTT and 1mM EDTA and this is frozen at -80 degC for refolding.

 

When ready to refold, I thaw the IB (room temp water) and then inject the IBs in two steps into 1M L-Arginine monohydrochloride, 10 mM TRIS-HCL, 50 mM NaCl pH 8.

A redox pair is present in the refold mix (10: 1 ratio GSG/GSSG) 

 

The refold volume is 400 mL so the Urea at this stage drops to around 200 mM.

 

The total protein added is approximately 80 mg.

This is refolded at 4 deg C, stirring overnight, the next day the solution is clear bar one or two stray floating particles which I get rid of by filtration

(solution is otherwise overall, clear). At this point I am happy as even with low urea the protein stays in solution, probably due to the L-Arginine.

 

I then proceed to dialysis against 10 mM TRIS-HCL, 50 mM NaCl pH 8.

The protein begins to crash out of solution until it looks like someone put fluffy white clouds in my dialysis bag.

 

What can I do? The protein is fine when the urea concentration is lowered by dilution in the refolding step but on dialysis it crashes!

 

Thank you in advance for the help!

-Luria Bertani-

you can try dialyzing in smaller arginine steps so that you don't shock your protein.

-mdfenko-

Thanks Mdfenko. The concentration 1M is high as is the volume of the dialysis (10L bucket), perhaps I can get the same effect by reducing the dialysis bucket volume?

-Luria Bertani-

you may still shock the protein. it would be better to prepare steps (you can work with smaller volumes of dialysis buffer).

-mdfenko-