Hi,
In a case like the one Raji outlined below - after all the attempts - I
would have most cerainly switched to insect cells as the next step :)
If you suspect that protein of interest has large disordered regions,
expression in a higher order system by itself may not be enough (still
good chances of proteolysis). I am guessing here, but co-expression with a
partner is probably necessary (as per your point #4). All of this assumes
that you need the entire protein, of course (as Raji said) - because
otherwise the fairly obvious next step could be to chop that beast into
manageable chunks :)
Is this 'normal' and 'expected'? I would say that yes, if you are working
with a large human protein that has potentially disordered regions and is
normally a part of a stable complex with other stuff - yes you can expect
problems exactly like these (or worse).
Artem
> BUT...I recently had a similar problem to what Mo outlines -- human
> protein in E. coli, 'zero induction', wondering if the protein is
> toxic based on colony morphology and effect on cell growth, and all
> the good stuff that Mo describes. In my case, for my human protein:
> 1. Disorder-prediction and secondary-structure-prediction programs
> indicate stretches of disordered protein
> 2. After the usual tricks of bacterial expression with a His-tagged
> version (temp, IPTG variation, autoinduction, cells), I got 'zero
> induction'. Then I switched to larger fusion tags like those both of
> you mention and then I could see expressed protein, though there was
> only a small fraction of full-length protein and copious amounts of
> proteolysis and/or truncated products (mass spec, Western blot etc.
> etc.).
> 3. When one tag didn't work, I switched to another. After switching a
> bunch of tags, I consistently faced the same kinds of problematic
> issues like persistent proteolytic degradation, persistent
> aggregation even when fusion tag was left intact, and hard-to-remove
> chaperone contamination during purification. I was unable to recover
> purified full-length protein and these issues could not be mitigated
> by buffer, pH optimization etc. during purification etc.
> 4. Literature indicates that the protein might function in a complex
>
> In my case, I used all of the above information collectively to
> hypothesize that my target protein, under the given conditions, was
> likely poorly folded on its own. In this specific case, even before I
> determined whether to move to a different expression system or not, I
> did still have at least a few choices that I can think of:
> a. To attempt co-expression with a protein binding partner
> b. To attempt co-expression in a chaperone-aided expression system
> c. To resort to a synthetic gene
> (Chopping up the protein and going after individual domains was not a
> real option in my case.)
>
> Is my example the norm or the exception? I leave that open.
>
> Even if my case is extremely rare, my original intention was to
> caution that there may come a time with a beast of a protein-- after
> a LOT has been attempted and tried-- when one has to start to quickly
> distinguish between a promising approach and a futile time-sink.
>
> To me, it seems we concur and there is not much scope for a healthy
> debate :)!
> Raji
>
>
>
>
> On Feb 26, 2009, at 10:07 PM, Artem Evdokimov wrote:
>
>> I personally think that debate is healthy.
>>
>> Having said this – I do not advertise one fusion partner over
>> another – and I did not intend to overweight the MBP success in my
>> previous message, it was just a familiar example. As I mentioned,
>> SUMO also worked for us – perhaps not as frequently as MBP but
>> there is no statistical significance in this because the number of
>> studied cases was way under a thousand (which intuitively sounds
>> like a useful number, but in fact even a thousand is probably too
>> small to be meaningful).
>>
>> Unlike choosing one brand of OS over another, the choice of fusion
>> partner (or indeed whether to use or not to use one) is not an
>> expensive one since cloning is generally very cheap and expression
>> in E. coli is also extremely affordable. Therefore it is perfectly
>> easy to try as many expression tricks as one desires. Having said
>> this I also should mention that enough trials in E. coli eventually
>> out-price a few trials in insect cells or other ‘higher order’
>> system so everyone should apply their own judgement as to when the
>> aggregate cost advantage disappears. I don’t think that anyone
>> would disagree that chances of successful expression of a
>> eukaryotic protein in e.g. insect cells are considerably higher
>> compared to the same odds in bacteria, but unfortunately so is the
>> price both in terms of money and of labor.
>>
>> Removal of fusion tags is a separate kettle of fish and certain
>> guidelines can be drawn from experience: viral proteases such as
>> TEV, TVMV, etc. are precise and somewhat slow (typical use ratio is
>> 1:10-1:50). Thrombin is very fast and furious (typical use ratio
>> 1:1000 to 1:5000) but it can sometimes cause undesired cutting if
>> you have either additional sites or dirty protease (it’s amazing
>> how much difference does thrombin purity make!). SUMO hydrolase
>> seems to be pretty fast but in my hands it stubbornly refused to
>> process quite a few protein fusions – which is probably about as
>> statistically significant as Stephen’s experiences to the opposite
>> J The menu of options is quite diverse so that everyone can find
>> something they like.
>>
>> So to recap – my key and only argument is that fusion with host
>> proteins (whatever they might be) is a valid and experimentally
>> successful method for salvaging expression of *some* difficult
>> proteins in bacterial hosts. The issues associated with this method
>> are also quite real – indeed, solubility enhancement is not equal
>> to folding enhancement but there is enough evidence in the field
>> that fusion proteins can provide more than just solubility and
>> brute yield enhancement. The experimenter has to be savvy enough to
>> figure out what’s going on quickly, not waste time trying to
>> optimize a hopeless case, and decide whether to proceed with more
>> trials or to go in a different direction.
>>
>> Artem
>>
>> ---
>> When the Weasel comes to give New Year's greetings to the Chickens
>> no good intentions are in his mind.
>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf
>> Of Stephen Weeks
>> Sent: Thursday, February 26, 2009 9:31 PM
>> To: [log in to unmask]
>> Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
>>
>> Hi all,
>> Once again I seem to have managed to kick up a minor debate on
>> the bulletin board (Note to self no more posts on SUMO or Apple :-
>> [ ). With quite a few years of experience working with SUMO I feel
>> I can safely state that it is a good enhancer of fusion protein
>> production in E. coli. I am personally NOT convinced that it is a
>> "solubility" enhancer like MBP or NusA but the fusions main benefit
>> is it's easy and specific removal. By default I do 24oC inductions
>> in Bill Studiers fantastic auto-inducing media so I haven't really
>> fought with solubility issues for a while. We make and use our own
>> hydrolase in the absence of any detergent (unless of course it the
>> target protein requires it) , and I find it better than TEV and
>> PreScission (admittedly I have not tried the new more soluble
>> clones of the former). Typically we get 100 mg/L using
>> autoinduction media which we dilute to 0.5 mg/ml in 50% glycerol
>> buffer, salt and DTT, of which I'll use 100-200 ul for a fusion
>> protein prep of 100 mg plus.
>> Addressing Mo's original question I shall restate my answer as:
>> that it would be cheaper to stick his construct into any fusion
>> vector he can lay his hands on before handing money over to the
>> gene synthesizers to see if he can get detectable expression.
>> Thinking downstream, if it works, you need to consider the expense
>> of the removal of the fusion partner. Clones are available for TEV,
>> PreScission (Rhinovirus 3c protease) and of course SUMO
>> hydrolase ;-) (plus there are few systems out there for removal
>> of tags without a protease). No single fusion system is a panacea
>> for all our protein expression woes and stating a position on one
>> is equivalent to choosing sides in the Mac vs. PC debate. (Actually
>> I have an idea for an advert featuring SUMO, the small and hip
>> fusion partner and MBP, the dull old and overweight workhorse).
>>
>> Stephen
>>
>>
>> [log in to unmask] wrote:
>> 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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