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] 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. 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
