Stabilizing overexpressed proteins
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Sun Feb 11 10:28:36 EST 2001
In article <SH0h6.274$v3.2224 at uchinews>, "Emir"
<ekhatipo at NOSPAMmidway.uchicago.edu> wrote:
:I actually had problems with concentrating the pure protein, too. It would
:precipitate at the concentration above ~5 mg/ml if dialyzed against 50%
:glycerol in the same buffer (50mM HEPES [can be replaced with Tris or Pi],
:200mM salt, etc.). I need the protein concentrated enough for further
:crystallization. The latter limits the number of compounds I would like to
:introduce into the buffer. High salt seems OK, same possibly with ethylene
:glycol (since it by itself can be used as a crystallization agent), but what
:about mild detergents (btw, thanks for reminding about good old cholate) and
:betaine? Would not they be a problem during crystallization? It is quite
:troublesome to get rid of them.
Ohhh, crystallization. The worst possible case... you never know
what's good and what's bad until you try. May or may not be a
problem. Betaine is frequent in many crystallization screens
(was _terrible_ for my protein; cholate had no effect and octyl
glucoside made crystals smaller - go figure), mild detergents
are also frequently tried but not recommended in the working
stock whenever possible. I can only say try increasing salt
first. A friend of mine was working on transposase and having
exact same problems (plus extreme cytotoxicity and poor solubility
in the cell). High salt solved solubility problem completely and
chitin binding/intein-based fusion expressed under severe repression
conditions with induction at 16C solved yields problems.
Crystallization part was actually relatively easy. The structure
was published in Science not long ago.
:Good weekend to everybody.
:"Dima Klenchin" <klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu> wrote in
:message news:961ti8$k78$1 at news.doit.wisc.edu...
:> "Emir" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote:
:> :I would appreciate someone recommending me a good strategy in finding
:> :conditions to prevent destabilization and aggregation of the recombinant
:> The good strategy is to take protein in stable form (high enough or low
:> enough salt, diluted enough, etc), and then shift it to conditions where
:> protein without "protectant" precipitates. Then you can easily see
:> effects of various additives.
:> :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
:> :strains used (pET11 in BLR cells, induction with lambda CE6), as
:> :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,
:> :NaCl, 1mM DTE (+protease inhibitor cocktail + PMSF).
:> First I would try increasing salt concentration to 0.5-1.0 M.
:> :However, once in solution, the protein tends to precipitate, as seen by
:> :increasing cloudiness of the extract, so that after IMAC on Talon resin
:> :elution of the protein with a 100mM imidazole step I end up with only
:> :of the protein, with the rest being not bound to resin, because of its
:> :aggregation. It could be that DNAse treatment destabilizes the protein,
:> :otherwise most of the protein will be pelleted along with the DNA during
:> :clearing of the cell lysate.
:> Again, rising salt might help here.
:> :I tried adding 10% glycerol to the buffer, sucrose, PEG 3600, but it did
:> :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,
:> Of these, my first choice would be betaine. Missing from the list
:> are mild detergents. In your case, I would first try cholate.
:> :If you had similar problems and succeeded in stabilizing your protein, I
:> We had solubility problems with one E.coli expressed PH domain -
:> it precipitated above certain concentration and precipitated whenever
:> frozen in tubes. The only solution we found was to keep it in 20%
:> ethylene glycol during purification, elute in large enough volume and
:> freeze by letting 25 ul drops go directly into jar with liquid nitrogen
:> way to aliquote, by the way - you can scoop resulting beads, place them
:> in cold eppendord and roll out one by one when necessary).
:> Good luck!
:> - Dima
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