I did crystallise a protein expressed in M. smegmatis a while ago (early 90's)! The cloning was done by Ying Zhang:https://www.jhsph.edu/faculty/directory/profile/786/ying-zhangIt might be worth dropping him a line. That's all I can suggest, sorry!!Good luck.Jon CooperOn 7 Jul 2020 10:07, Matthew
Hi all,
If anybody is interested in non-viral stable expression, we have a
piggybac-based, doxycycline-inducible system. It is the reference 40 in the
lentiviral paper that Tomas directed to. We’d be happy to distribute the
plasmids.
Zhijie
> On Jan 25, 2020, at 3:26 AM, Tomas Malinauskas
Hi Gloria,
two key papers describing expression (transient and lentivirus-based)
of proteins in HEK293 cells we use to make milligrams of proteins for
crystallization and cryo-EM:
Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1243-50. Epub
2006 Sep 19.
A time- and cost-efficient system
7:10:06 AM
To: David Briggs ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] protein expression in human cells
Dear colleagues,
I wonder if there were a bit less controversial possibility. No matter if that
was less efficient. Would there be an option of using human cell lines that do
not origin f
:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein expression in human cells
Hi Gloria,
Another vote here for HEK 293 Expi or Freestyle.
The off-the-shelf transfection reagents are super expensive, but I make my own
(happy to share protocols with anyone interested) and we normally get
January 25, 2020 1:31:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein expression in human cells
In our lab, we see Expi293F works very good.
Thanks,
Reza
Md. Rezaul Karim
Graduate student, PhD Program in Integrated Biomedical Sciences
Morsani College of Medicine
University of South
Hi,
I’ve received a number of concurring suggestions. Some have requested more
detail about the experiments. Here are the details.
1. Cells: BL21(DE3)
2. Plasmid: pET28a (T7 promoter)
3. Media: TB
4. Protein: cytoplasmic
5. Expression: Grow at 37degC until OD600=0.6, cool to 20degC, induce with
Hi Reza,
A few month ago I had a the exact same problem and we checked everything
we could think of but without any improvement. Finally we were able to
solve the problem only by subcloning the ORF into another plasmid (pET9a
or pET29b). The big difference being the His tag position (C-ter or
: Friday, March 20, 2015 8:06 AM
To: Reza Khayat
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein expression
Reza,
someone might have mentioned this, it takes longer time to cool down larger
culture, we turn down the temp of the shaker when OD is about 0.4. For some
protocol, we even use ice
Reza,
someone might have mentioned this, it takes longer time to cool down larger
culture, we turn down the temp of the shaker when OD is about 0.4. For some
protocol, we even use ice to cool the flask down before induction. You
might also want to consider a lower induction temp, like 16degC.
Hi Reza,
In addition to the many useful suggestions already made, I would suggest
lowering the final concentrations of IPTG. In many cases, 1mM IPTG
interferes with expression levels and/or solubility. This suggestion does
not address your concern for why things become ugly in going from 3mL to
Dear Reza,
It sounds to me like an aeration issue. I don't of course know how the 3 ml
culture is grown, but if say the small culture is less perfectly aerated
and slightly anaerobic, slower growth would mean slower metabolism and
slower protein production as well so things do not build up so
Question: you're taking a 3-ml equivalent out of 500 ml culture, and
processing it as if it was a 3ml culture? Or are you basing the result on
processing the entire 500ml?
Reason I ask this is simply to make sure your extraction/purification
timing is the same in both cases. It matters!
Assuming
Hi Reza.
Clearly nobody needs to know anything about what protein you are
specifically working on; that being said, in order to avoid a potentially
endless email string of expert advices, please include everything
detail-wise regarding your expression system, culture conditions,
induction, and
If you are eligible, I'd highly recommend the EMBO courses in Europe or the
CSHL courses in the US on those subjects. Reading is a good start but will
not be enough if you want to do real work.for strategy considerations, there
is always Ch 3 and 4 in le livre.
Best, BR
From: CCP4 bulletin
Hi Chen,
In this case, it seems that linker region is of great importance for the
proper folding of the two linked domains. I have not much experience in
linker region design, generally use (GS)5-10 times. However it depends on
individual case. Anyone who has successful experience in linker
I have been trying to express a rat protein in bacteria. The MBP-fusion
expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag
only gave inclusion bodies. The problem is that all protein runs in the void
volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl),
I wholly agree with the below. I am not sure how well E.coli can correctly fold
snaky
misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out
that tags do it
sometimes...
Folded by association for insoluble proteins has often not worked well for
me. Sometimes, when it
Chen and David,
Before adding detergent, be forewarned that the MPB in many fusions
will not bind to an amylose column in the presence of most
detergents, particularly maltoside detergents. It has been the bane
to us so we have engineered MBP vectors with His tags to deal with
this.
Hi Chen,
Since you recognize that this is a protein expression problem, the best way
to get return of your investment of efforts is to get soluble expression
instead of trying to solubilize the protein down stream.
Many good suggestions have been proposed. I just want to add one more thing
Hi Chen,
You could try adding some detergent or other solubilising agent (eg
NDSBs) to your buffer.
Have you tried other pHs? If you are sat near to or on the pI of your
protein, it will be at its least soluble and more likely to aggregate.
I've had protein behave like yours at pH 7.5 but behave
I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 on
minimal media, but in my case I did get protein expression. The way I got
around it was as follows. I grew the cells up in minimal media plus the
amino acids that suppress methionine biosynthesis, plus L-methionine.
Manish,
I also had a similar problem getting cells (C41 (DE3) in my case) to grow
in minimal media. To get around this problem, I took cells from an agar
plate and grew them in a small volume (5 mL) of the minimal media. Once
that culture got thick, I then inoculated 200 mL of minimal media with
Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin
requiring and can not grow in the absence of thiamin. The thiamin
requirement is so low that you can often get slow growth to a low OD
based on residual thiamin in the cells, but you will not get robust
growth.
Also,
Manish,
Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...
M9 medium can be slightly difficult. However, no one says that you *have*
to use minimal medium such as M9 (again, assuming that you're doing
: Thursday, April 19, 2007 10:31 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish,
Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...
M9 medium can
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