Dear colleagues
The following position is available in Grenoble (France):
Protein Expression and Purification Scientist, Biochemist, Structural Biologist
Job Description
A research technician position is available in the group Structural Biology of
Novel Drug Targets in Human Diseases (https://
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 S
Hi all
I'm designing genes for expression in M. smegmatis (safe host for Mtb
proteins), but its not possible (or advisable due to the GC content) to
optimise for
mycobacterial expression.
Would anyone with experience be able to tell me if its fine to stick with the
E. coli codon optimisation,
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
, 2020 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
no
: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
In our lab, we see Expi293F works very good.
Thanks,
Reza
Md. Rezaul Karim
Graduate student, PhD Program in Integrated Biomedical SciencesMorsani College
of Medicine
University of South Florida
E-mail: reza...@yahoo.com, rez...@health.usf.eduPhone: +1-954-937-8487
On Friday, January 24, 202
Hello CCP4-ers,
I was wondering what people have found to be the best human cell line
expression system for making a large quantity of purified recombinant
protein.
Any information and protocols would be greatly appreciated.
Happy 2020, Gloria
##
Dear All,
There is an opportunity to join the Protein Biochemistry team at Vertex
Pharmaceuticals based in the UK as a Research Scientist (Protein expression and
purification). Please see the advert in the link below. Interested candidates
should apply directly through our website. Please do no
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
50
If you try to lyse a cell pellet from 500 ml via sonication compared to the 3
ml that is a huge difference. In particular if you don't scale up the total
volume. Say you lyse your 3 ml culture in 10 ml and your 500 ml in 20 ml. Your
local protein concentration is perhaps 50x higher then, which m
.
Vaheh Oganesyan
www.medimmune.com
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Artem
Evdokimov
Sent: Thursday, March 19, 2015 6:25 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein expression
Question: you're taking a 3-ml equivalent out of 500 ml cu
: 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
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
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. Maybe
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.
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 lysi
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
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 easi
Hi,
We can express quite a bit of soluble protein when growing 3ml cultures.
However, the protein becomes insoluble (inclusion bodies) when we scale up to
500ml cultures. Has anyone experienced such a problem, and found a solution to
it? Thanks.
Best wishes,
Reza
Reza Khayat, PhD
Assistant Pr
letin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hena
Dutta
Sent: Thursday, July 21, 2011 10:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein expression, purification and crystallization
Dear Members of CCP4BB and PHENIXBB,
Can you suggest a reliable website where I can get basi
Dear Members of CCP4BB and PHENIXBB,
Can you suggest a reliable website where I can get basic and advanced
information on protein expression, purification and crystallization. I like
to read on the monitor.
Thanking you in advance...
Hena
Associate Director Protein Expression Group, Novartis Institutes for
Biomedical Research, Basel, Switzerland
Job description:
As Group Leader in the Structural Biology Platform (SBP) within the Center
for Proteomic Chemistry you will lead a group dedicated to all aspects of
protein expression i
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 region
Hi,
I have a protein with two independently folded domains. I can express either
one in bacteria with pretty good expression yield. However, when I put them
together with a linker, the expression drops significantly. I can barely see
any soluble protein and most of it is now inclusion bodi
10:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] protein expression problem
Hi,
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
Another point not yet brought up is how do you express ?
things to check:
- different media (LB. TB, auto)
- when do you induce (mid log phase, or end log phase)
- how long do you induce (before reaching stationary phase, going over
night into starvation phase)
- how much IPTG do you add
- MBP
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. Wha
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
'
> 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
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 p
Hi,
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
: 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 be
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
Se-Me
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, min
://crystal.umaryland.edu
>
>
> -Original Message-
> From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
> [EMAIL PROTECTED]
> Sent: Wednesday, April 18, 2007 10:34 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Protein expression in Minimal media (M9)
&g
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 0
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: Wednesday, April 18, 2007 10:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein expression in Minimal media (M9)
Hello everybody,
Sorry for an offtopic question. I am trying to express a protei
Hi,
from my experience with minimal media expression you're probably
better off starting with a good, thick rich media overnight growth,
and then diluting that out 1:20 – 1:50 into the M9.
Richard
--
Richard P. Grant
School of Molecular & Microbial Biosciences University of Sydney
Hello everybody,
Sorry for an offtopic question. I am trying to express a protein in M9
minimal media for Selenomet incorporation. When grown in LB this protein
expressed very well and got good crystals. Diffraction was upto 2 A. I am
having a hard time expressing the same protein in Minimal me
The original e-mail was sent with the wrong subject heading. This is
NOT a Post-Doctoral position, and a PhD will not be required.
Protein Expression Research Technologist
The newly established structural virology lab within the Molecular
Virology and Vaccines Branch of the Influenza Divisio
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