Stabilizing overexpressed proteins

Michael Witty mw132 at mole.bio.cam.ac.uk
Sun Feb 11 06:54:44 EST 2001


I recently had a similar problem with protein precipitation, which I
solved by adding 0.1% detergent (Triton X100, Tween20 and NP40 all worked
for me).  However, I have not tried an assay for activity yet though.
Mike.

On Fri, 9 Feb 2001, Emir wrote:

> Hi,
> I would appreciate someone recommending me a good strategy in finding
> conditions to prevent destabilization and aggregation of the recombinant
> proteins.
> 
> I can express my 6-His tagged DNA-binding recombinant protein (RecA
> homologue) in the range of 5 to 50 mg/l, depending on conditions and E.coli
> strains used (pET11 in BLR cells, induction with lambda CE6), as estimated
> by Westerns with cell lysates. I can extract most of the protein by
> freeze-thawing, DNAse treatment, and sonication in 50mM HEPES, pH 7.5, 200mM
> NaCl, 1mM DTE (+protease inhibitor cocktail + PMSF).
> 
> However, once in solution, the protein tends to precipitate, as seen by
> increasing cloudiness of the extract, so that after IMAC on Talon resin and
> elution of the protein with a 100mM imidazole step I end up with only ~10%
> of the protein, with the rest being not bound to resin, because of its
> aggregation. It could be that DNAse treatment destabilizes the protein, but
> otherwise most of the protein will be pelleted along with the DNA during the
> clearing of the cell lysate.
> 
> I tried adding 10% glycerol to the buffer, sucrose, PEG 3600, but it did not
> stabilize the protein.
> Any ideas on what compounds to try to stabilize the protein? One of the
> Creighton's lab manuals recommends PEG 1000, guanidine sulfate (not
> chloride), betaines, TMAO, MPD (which is quite expensive), (NH4)2SO4, MgSO4,
> etc.
> 
> If you had similar problems and succeeded in stabilizing your protein, I
> would appreciate your sharing your experience here.
> Thank you.
> 
> Emir
> 
> 
> 
> 






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