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Hi,
I respectfully disagree with the doom&gloom feelings regarding fusion
proteins. In my not very limited experience, fusion proteins *can* fix
expression issues. Do they always work - of course not :) But there are
very few things in this field that work most of the time. Is it better to
try a fusion protein or to go into a higher-order expression system? If
you can afford it, usually higher order systems tend to work better. But
what if you cannot afford it?
Regarding precipitation upon cleavage - consider the example of PTPbeta
catalytic domain: this protein expresses very poorly on its own, however
it expresses extremely well with a His-MBP N-terminal fusion, and the
activity of the fusion protein is very high. If you cleave the protein in
'just buffer' then PTPbeta rapidly precipitates. Bad news, right? However
if you cleave the fusion in the presence of 0.1% BOG the protein stays
perfectly soluble and monomeric, concentrates to 15 mg/ml and produces
marvellous crystals (about six structures in the PDB). So - do not be too
quick to dismiss fusion proteins as a way to try and salvage your
desperate cases, especially if going to a different expression system is
hard for some reason.
Regarding SUMO - I have personally tested it on about 30-35 proteins. It
only worked for *one* - but it made the protein nice and soluble, and it
stayed soluble after cleavage (note - we do not use the SUMO-protease,
just regular protease sites).
Is ratio like that worht the trouble? You decide :)
Artem
> Some thoughts about SUMO tags and fusion tags in general.
>
> Fusion tags also follow the "Garbage In, Garbage Out" philosophy.
> Yes, if for many of the reasons already hashed out extensively on
> CCP4BB, one is dealing with lack of expression or miniscule
> expression, often tagging the protein with a fusion/cleavable tag
> does indeed bump up the expression and lead to 'improved solubility'.
> Sometimes, it's very important to ask: improved solubility of what
> though?
>
> Everything that Phoebe describes, namely the chaperone contamination,
> precipitation after cutting off tag etc., reeks of an intrinsically
> misfolded/unstable/unhappy protein. My experience-- and those of many
> others-- is that the fusion tag and fusion tag alone can only fix
> little in cases: 1) when one observes lots of degradation of the
> untagged protein, 2) where the untagged protein is made as an
> intrinsically misfolded/unstable protein. In these cases, the carrier
> protein then notoriously comes along for the ride in the soluble
> fraction with the fusion/cleavable tag, initially giving the
> impression of improved expression and improved solubility. Even then,
> one might even see multiple degradation products with the tagged
> expression product. Next, cleave the tag off in such a case and lo
> and behold! all protein precipitates and you are back to square one.
>
> I am not trying to discourage anyone from using fusion tags -- to
> improve expression, solubility, crystallization etc. We all know of
> many examples where fusion tags have worked wonders. I only caution
> that if your favourite protein is intrinsically misfolded in a
> particular expression system and then you have tried tagging a fusion/
> cleavable tag onto the protein in the same expression system and you
> observe all that Phoebe describes, perhaps it is time to bang your
> head against a different wall now. In many difficult cases, I am
> unaware that a fusion tag actually aids in the proper folding of a
> carrier protein. I will not rule out this possibility but I do not
> know that this is the general rule.
>
> I have worked quite a bit with SUMO tags. As far as GST and SUMO tags
> are concerned, I banged my head against the GST-tag and SUMO- tag
> wall for my target protein for a frustrating while. I tried a His
> tag, then a GST tag, then a SUMO tag. All had exactly the same
> symptoms. In my case, clearly the problem lay with the carrier
> problem but I was never allowed to conclude so.
>
> Just my two cents, the worth of which will already have diminished by
> the time you have read this email.
>
> Raji
>
>
>
>
>
>
> On Feb 26, 2009, at 11:30 AM, Phoebe Rice wrote:
>
>> We haven't tried SUMO, but had some frustrating results with
>> GST fusions. They did improve expression and solubility - BUT
>> in one case the target protein precipitated immediately when
>> the tag was cleaved off, and resisted all attempts to bring it
>> back to life. In another case, the fusion protein dragged
>> chaperones into the prep that were nearly impossible to get
>> rid of completely, thus ruining our ATPase assays.
>>
>> Is SUMO, being smaller, less likely to drag such crud along
>> with it?
>>
>> Phoebe
>>
>>
>> ---- Original message ----
>>> Date: Wed, 25 Feb 2009 14:48:57 -0500
>>> From: Mo Wong <[log in to unmask]>
>>> Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
>> E. coli
>>> To: [log in to unmask]
>>>
>>> Thanks to all who responded. Actually, this bulletin
>>> board is better for help with molecular biology than
>>> the molecular biology bulletin board I am subscribed
>>> to!
>>>
>>> On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks
>>> <[log in to unmask]> wrote:
>>>
>>> Mo,
>>> Just to add my 50 cents, I didn't see any
>>> mention of the use of fusion proteins in your
>>> original post. GST, MBP or my personal, and
>>> completely biased, favourite SUMO (plus many more
>>> proteins) have been shown to enhance expression
>>> when fused to the amino terminus of a target
>>> protein. If you fear you have toxicity, simply
>>> tracking the OD600 pre and post induction normally
>>> tell you if this is happening. I've worked with
>>> proteins that basically baselined the cell growth
>>> upon induction and, as Artem stated, at least I
>>> knew my protein was being made albeit at very low
>>> levels.
>>>
>>> Stephen
>>>
>>> --
>>> Stephen Weeks, Ph. D.
>>> Drexel University College of Medicine
>>> Department of Biochemistry and Molecular Biology
>>> Room 10102 New College Building
>>> 245 N. 15th St.
>>> Philadelphia, PA 19102
>>>
>>> Phone: (+) 215-762-7316
>>> Fax: (+) 215-762-4452
>>>
>>> Mo Wong wrote:
>>>
>>> I thought I'd post this to the CCP4bb, as
>>> judging by previous posts, it seems I could get
>>> some useful insight into my problem...
>>>
>>> This is question has probably been asked by
>>> people for a long as molecular biology has been
>>> around, but hopefully my question isn't a
>>> complete rehash of other peoples: I am trying to
>>> express a human protein in bacteria where the
>>> only modified amino acids are 3 phosphorylated
>>> serines. I’ve gone through the usual hoopla of
>>> trying to get it expressed in E. coli
>>> (Rosetta/Codon+ cells, varying IPTG, low
>>> temperature, etc). Sequencing confirms my insert
>>> is correct, but from coomassie gel inspection, I
>>> appear to get near zero induction (I need to do
>>> a Western to get a clearer assessment). I’ve
>>> heard about custom gene synthesis, and it
>>> appears Mr. Gene (https://www.mrgene.com/) would
>>> be a good avenue to look into as they optimize
>>> the ORF taking into account codon usage in E.
>>> coli (though I’m not sure they examine
>>> putative mRNA substructure formation like some
>>> companies do). It’s only 49c per base pair, so
>>> doesn’t seem too cost prohibitive. My only
>>> concern is that if this protein is toxic, I
>>> could be wasting money.
>>>
>>> So I was wondering, has anyone seen the
>>> expression for a particular protein change from
>>> zero in Rosetta/Codon+ cells using "native"
>>> sequeneces to being largely overexpressed in
>>> BL21(DE3) cells using codon optimized sequences?
>>> For folks who have had a similar problem to the
>>> one I've described, would you recommend that I
>>> first try using a codon optimized sequence in E.
>>> coli over testing protein expression in
>>> yeast/insect cells, or the other way round?
>>>
>>> Thanks!
>> Phoebe A. Rice
>> Assoc. Prof., Dept. of Biochemistry & Molecular Biology
>> The University of Chicago
>> phone 773 834 1723
>> http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/
>> 01_Faculty_Alphabetically.php?faculty_id=123
>>
>> RNA is really nifty
>> DNA is over fifty
>> We have put them
>> both in one book
>> Please do take a
>> really good look
>> http://www.rsc.org/shop/books/2008/9780854042722.asp
>
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