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Just in case anyone else encounters the same problem, like Artem I have had a few SUMO fusion constructs that have stubbornly refused to cleave even with a 1mg of enzyme (Ratio ~ 1:100) at 37c in the presence of low concentrations of chaotrope. In all cases the problem was solved by inserting a glycine residue between the cleavage site and the first amino acid (always a methionine in my case). This resulted in same constructs being able to be cleaved at 4oC, with a Ratio 1:1000 to 1:2000 of hydrolase in 30 minutes (Cleavage was performed in a tyipcal IMAC elution buffer with 250 mM NaCl). Of course by doing this you will no longer have the "Native" amino terminus on you protein but funnily enough have the same additional residue that TEV leaves behind. I perform all my cloning using LIC but if I remember correctly the NYSGC use a BamHI (GGATTC) for cloning downstream of the SUMO cleavage site which will introduce an additional serine residue. I assume, without ever seeing their cleavage data, that they never had a cleavage problem. In the one case where I finally got crystals of the protein that initially poorly cleaved as a SUMO fusion construct the amino terminus was highly ordered in the structure (I could use the Selenium labeled start methionine for phasing). I'm curious if this can be extrapolated to all poorly cleavable fusion constructs. 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 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] <mailto:[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]> <mailto:[log in to unmask]> >>>> Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in >>>> >>> E. coli >>> >>>> To: [log in to unmask] <mailto:[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]> <mailto:[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 >>> > > >