protein degradation

From: Sivasankar Putta
Date: 15 February 2012 13:09

Dear All,

Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli.  The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT  throughout during purification ( right from lysis stage).   We handle the protein at 4 degree Centigrade.

Can you please suggest what precautions we can try to avoid such degradation ?

   

       Please find the attached gel picture regarding protein

Sivasankar Putta

  

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From: Roger Rowlett
Alternatively, why not do a quick polish step using a long gradient on a 5 mL anion or cation exchange column, followed by a gel exclusion cleanup if necessary? Once the proteases have been removed from the crude extract, further degradation of purified protein should be minimal. This purification treatment routinely works for us with proteins at this contaminant level.

Roger Rowlett

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From: Bosch, Juergen

Late induction for short time. Then immediately purify it cut down on any unnecessary steps eg shorter spin all on ice or coldroom etc.

Jürgen

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Jürgen Bosch

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From: Christian Roth
Hi,
you may also check things like chemical degradation in SDS buffer as part of
the analysis. Esspecially your degradation pattern is very much constant
throughout your whole purification procedure.

Christian

Am Mittwoch 15 Februar 2012 14:09:19 schrieb Sivasankar Putta:
> in* E. Coli.*  The protein appears to degrade during purification; we have

--
Christian Roth

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From: Mark J van Raaij
try experimenting with different, especially protease-deficient, E coli strains to express the protein and try different methods to lyse the bacteria (sonication, french-press, emulsification, bead-beater, mortar & pestle under liquid nitrogen).

on the other hand, if you are lucky, you are just proteolysing some surface loops and can still purify and crystallise the protein. This was done on purpose for the cap-binding complex, see:
Crystal structure of the human nuclear cap binding complex.
Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S.
Mol Cell. 2001 Aug;8(2):383-96.

Mark J van Raaij

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From: Xiaodi Yu

Hi Sivasankar:

Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein.
After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and  small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration).
One small "trick" you can try is wash the cell with the buffer containing PMSF once before lysising the cell.

Yu Xiaodi

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From: Jacob Keller 

You can also try putting a different affinity tag on the other
terminus, and use that as a second step.

JPK

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*******************************************
Jacob Pearson Keller

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From: Carlos Kikuti
I agree with Mark, except that I wouldn't even try sonication, Triton or freeze/thaw cycles in that case.

I'd look for emulsification (with a Homogenizer) in a cold room, but if you go quickly and gently with the French Press (either in a cold room or by using a cold piston) it might help. Don't use too much pressure, it heats up the sample.

I also agree that if it migrates as a single peak in gel filtration and in heparin sepharose, there is no reason for not setting some drops with it. And if you decide to do it, then simplify the purification and avoid submitting the protein to treatments that are not helping to get it purer.

(I just found it weird that your "fraction 6" has a huge load of protein , I guess those are actually the beads from the purification or something like that? In any case it seems to me that the fraction volume could be increased)

Carlos