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:[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] <mailto:[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]> <mailto:[email protected]>
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in
E. coli
To: [email protected] <mailto:[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]> <mailto:[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. 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
