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Protein Refolding - Precipitation during dialysis

Refolding Protein

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#1 Luria Bertani

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Posted 26 May 2014 - 12:06 AM

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!


Edited by Luria Bertani, 26 May 2014 - 02:43 AM.


#2 mdfenko

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Posted 27 May 2014 - 04:19 AM

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


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#3 Luria Bertani

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Posted 27 May 2014 - 04:43 AM

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?



#4 mdfenko

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Posted 27 May 2014 - 05:20 AM

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


talent does what it can
genius does what it must
i do what i get paid to do




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