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 dont 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 (its 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 Stephens 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 whats 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:[email protected]] On Behalf >> Of Stephen Weeks >> Sent: Thursday, February 26, 2009 9:31 PM >> To: [email protected] >> 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 >> >> >> [email protected] 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 <[email protected]> >>>>> Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in >>>>> >>>> E. coli >>>> >>>>> To: [email protected] >>>>> >>>>> 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 >>>>> <[email protected]> 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. Ive 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). Ive >>>>> 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 Im not sure they examine >>>>> putative mRNA substructure formation like some >>>>> companies do). Its only 49c per base pair, so >>>>> doesnt 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 >>>> >> >> >> > >
