[ccp4bb] Open Post-Doc and technician positions

2009-02-24 Thread Peer Mittl 

Applications are invited for four positions at various levels at the
University of Zürich (Switzerland). Currently there are four vacant
positions at the postdoctoral and research assistant levels (2 Post-Docs
and 2 research assistants) in the lab of Prof. Markus Grütter in the
Department of Biochemistry. We are focusing on the investigation of the
structure/function relationship of proteins involved in apoptosis,
innate immunity and trans-membrane signaling (PNAS (2008) 105:20251,
Structure (2008) 16:1443, Structure (2007) 15:625, PLoS Biol (2007) 5:e7).

We seek enthusiastic team players with experience in at least one of the
following disciplines: protein X-ray crystallography, protein expression
and purification, and eukaryotic expression systems. Experiences in
membrane protein biochemistry will be an advantage. Good knowledge of
English is mandatory. Candidates applying for the Post-Doc position
should hold a PhD in a life science-related subject. All positions are
time limited for three years. Salary will be according to the
regulations of the Swiss National Science Foundation scheme.

Please submit your electronic application including a CV, a list of
publications (for the Post-Doc positions) and the names of two to three
referees to Mrs. Salome Rittmeyer at the following address:
secgruet...@bioc.uzh.ch

Best regards,
Peer Mittl




vacantPosition_UZH.pdf
Description: Binary data

[ccp4bb] Post-doc position available

2009-02-24 Thread Van Den Berg, Bert 
A postdoctoral position is available in the laboratory of Bert van den Berg at 
UMass Medical School, Worcester (MA), to work on a NIH-funded project 
investigating the mechanism by which hydrophobic molecules, such as toxic 
xenobiotics, cross the bacterial outer membrane. The focus of the project will 
be on establishing in vivo transport assays for (mono) aromatic hydrocarbons, 
and the purification, crystallization and structure determination of FadL 
homologues from biodegrading bacteria. In addition, channel-substrate complexes 
and site-directed mutants of the toluene channels TodX and TbuX will be 
subjected to structural studies. For more information please see Hearn et al., 
Nature 1 Feb 2009, and Hearn et al., PNAS 105, 8601-8606 (2008), or contact me 
directly.
 
We are looking for a dedicated and enthusiastic person with a strong background 
in molecular biology. Experience with cloning in Pseudomonas species would be 
ideal. Experience in membrane protein biochemistry and X-ray crystallography 
would be advantageous, but is not required. Please send applications and the 
names of two or three references to: bert.vandenb...@umassmed.edu.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Peer Mittl
Sent: Tue 2/24/2009 7:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Open Post-Doc and technician positions



Applications are invited for four positions at various levels at the
University of Zürich (Switzerland). Currently there are four vacant
positions at the postdoctoral and research assistant levels (2 Post-Docs
and 2 research assistants) in the lab of Prof. Markus Grütter in the
Department of Biochemistry. We are focusing on the investigation of the
structure/function relationship of proteins involved in apoptosis,
innate immunity and trans-membrane signaling (PNAS (2008) 105:20251,
Structure (2008) 16:1443, Structure (2007) 15:625, PLoS Biol (2007) 5:e7).

We seek enthusiastic team players with experience in at least one of the
following disciplines: protein X-ray crystallography, protein expression
and purification, and eukaryotic expression systems. Experiences in
membrane protein biochemistry will be an advantage. Good knowledge of
English is mandatory. Candidates applying for the Post-Doc position
should hold a PhD in a life science-related subject. All positions are
time limited for three years. Salary will be according to the
regulations of the Swiss National Science Foundation scheme.

Please submit your electronic application including a CV, a list of
publications (for the Post-Doc positions) and the names of two to three
referees to Mrs. Salome Rittmeyer at the following address:
secgruet...@bioc.uzh.ch

Best regards,
Peer Mittl

Re: [ccp4bb] TLS refinement in Refmac gets stuck

2009-02-24 Thread Anita Lewit-Bentley 

Hi Ian,

A bug was reported with that version of Refmac, though I've no idea  
if that would cause your problem: you should upgrade to the latest  
version from the York website.


Done - and here is what happens:

1) the latest CCP4 package (6.1.1) runs the same version of Refmac  
(5.5.0066) and, predictably, crashes the same way;
2) on the Refmac webside there are much more recent versions of  
Refmac, not yet incorporated into the CCP4 package. Versions 72 and  
higher have a bug fix for TLS refinement.
3) included version 5.5.0086 (the precompiled version for Mac - intel)  
in CCP4  6.1 - and that runs fine 


except that it exits with a "bus error, child killed"

I HATE seeing a child killed 

but anyway, the output pdb and mtz files exist in the "scratch"  
directory with a .tmp extension, but otherwise perfectly useable.


So I can scrape along if I remember to copy the .tmp files accross to  
my working directory. Better than nothing!


Thanks for your hint.

Cheers,

Anita




-Original Message-
From: owner-ccp...@jiscmail.ac.uk [mailto:owner- 
ccp...@jiscmail.ac.uk] On

Behalf Of Anita Lewit-Bentley
Sent: 23 February 2009 15:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: TLS refinement in Refmac gets stuck

Dear ccp4 folks,

I am refining a structure in several crystal forms, with several  
molecules
in the asymmetric unit. The structure is rather flexible, so I am  
using
TLS refinement, having first submitted the coordinates to the TLSMD  
server

(http://skuld.bmsc.washington.edu/~tlsmd/
 ).

The TLS refinement worked OK with REFMAC 5.2.0019 (CCP4 6.0.2) for 3
molecules in the asu, but not for crystal forms with 6 molecules/ 
asu (it

just got hung).

For the latter crystal forms I have switched to CCP4 6.1.0 with  
REFMAC
5.5.0066, where it worked fine for two crystals - and now plays up  
for the

third crystal!

It is not exactly crashing, so I don't know what is going on. This  
is the

last part of the logfile:

"Problem
xyz 1230   5.6147661  -5.1691742   2.8234024 -
1.55429840E-02  9.35771316E-02  0.17803398  0.23723726  
-0.13626081
5.39243035E-02 -0.14011805 -1.55429840E-02  0.11864197   
0.40574944

0.71992165  0.57164854 -0.39361250
**
*
* Information from CCP4Interface script
**
*
Writing final coordinates (XYZOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.pdb
**
*

**
*
* Information from CCP4Interface script
**
*
Writing final phases (HKLOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3M_Se_TLS_refmac1.mtz
**
*

**
*
* Information from CCP4Interface script
**
*
Writing final TLS (TLSOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.tls
**
*
"
Unfortunately, there are NO files written out! There is no real error
message either, the CCP4i interface says the job has finished  
normally.


I have already tried to vary the number of TLS fragments, with no  
better

result. The older Refmac just gets hung

Any help will be appreciated!

Thanks,

Anita


Anita Lewit-Bentley

Unité d'Immunologie Structurale
CNRS URA 2185
Département de Biologie Structurale & Chimie
Institut Pasteur
25 rue du Dr. Roux
75724 Paris cedex 15
FRANCE

Tel: 33- (0)1 45 68 88 95
FAX: 33-(0)1 40 61 30 74
email: ale...@pasteur.fr





Disclaimer
This communication is confidential and may contain privileged  
information intended solely for the named addressee(s). It may not  
be used or disclosed except for the purpose for which it has been  
sent. If you are not the intended recipient you must not review,  
use, disclose, copy, distribute or take any action in reliance upon  
it. If you have received this communication in error, please notify  
Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com  
and destroy all copies of the message and any attached documents.
Astex Therapeutics Ltd monitors, controls and protects all its  
messaging traffic in compliance with its corporate email policy. The  
Company accepts no liability or responsibility for any onward  
transmission or use of emails and attachments having left the Astex  
Therapeutics domain.  Unless expressly stated, opinions in this  
message are those of the individual sender and not of Astex  
Therapeutics Ltd. The recipi

Re: [ccp4bb] TLS refinement in Refmac gets stuck

2009-02-24 Thread Eleanor Dodson 
Yes - I found that irritating bug; it is a disaster for less experienced 
users..


It doesnt seem to happen with the linux installation..

 Eleanor


Anita Lewit-Bentley wrote:

Hi Ian,

A bug was reported with that version of Refmac, though I've no idea 
if that would cause your problem: you should upgrade to the latest 
version from the York website.


Done - and here is what happens:

1) the latest CCP4 package (6.1.1) runs the same version of Refmac 
(5.5.0066) and, predictably, crashes the same way;
2) on the Refmac webside there are much more recent versions of 
Refmac, not yet incorporated into the CCP4 package. Versions 72 and 
higher have a bug fix for TLS refinement.
3) included version 5.5.0086 (the precompiled version for Mac - intel) 
in CCP4  6.1 - and that runs fine 


except that it exits with a "bus error, child killed"

I HATE seeing a child killed 

but anyway, the output pdb and mtz files exist in the "scratch" 
directory with a .tmp extension, but otherwise perfectly useable.


So I can scrape along if I remember to copy the .tmp files accross to 
my working directory. Better than nothing!


Thanks for your hint.

Cheers,

Anita




-Original Message-
From: owner-ccp...@jiscmail.ac.uk 
[mailto:owner-ccp...@jiscmail.ac.uk] On

Behalf Of Anita Lewit-Bentley
Sent: 23 February 2009 15:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: TLS refinement in Refmac gets stuck

Dear ccp4 folks,

I am refining a structure in several crystal forms, with several 
molecules

in the asymmetric unit. The structure is rather flexible, so I am using
TLS refinement, having first submitted the coordinates to the TLSMD 
server

(http://skuld.bmsc.washington.edu/~tlsmd/
 ).

The TLS refinement worked OK with REFMAC 5.2.0019 (CCP4 6.0.2) for 3
molecules in the asu, but not for crystal forms with 6 molecules/asu 
(it

just got hung).

For the latter crystal forms I have switched to CCP4 6.1.0 with REFMAC
5.5.0066, where it worked fine for two crystals - and now plays up 
for the

third crystal!

It is not exactly crashing, so I don't know what is going on. This 
is the

last part of the logfile:

"Problem
xyz 1230   5.6147661  -5.1691742   2.8234024 -
1.55429840E-02  9.35771316E-02  0.17803398  0.23723726 
-0.13626081
5.39243035E-02 -0.14011805 -1.55429840E-02  0.11864197  
0.40574944

0.71992165  0.57164854 -0.39361250
** 


*
* Information from CCP4Interface script
** 


*
Writing final coordinates (XYZOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.pdb
** 


*

** 


*
* Information from CCP4Interface script
** 


*
Writing final phases (HKLOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3M_Se_TLS_refmac1.mtz
** 


*

** 


*
* Information from CCP4Interface script
** 


*
Writing final TLS (TLSOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.tls
** 


*
"
Unfortunately, there are NO files written out! There is no real error
message either, the CCP4i interface says the job has finished normally.

I have already tried to vary the number of TLS fragments, with no 
better

result. The older Refmac just gets hung

Any help will be appreciated!

Thanks,

Anita


Anita Lewit-Bentley

Unité d'Immunologie Structurale
CNRS URA 2185
Département de Biologie Structurale & Chimie
Institut Pasteur
25 rue du Dr. Roux
75724 Paris cedex 15
FRANCE

Tel: 33- (0)1 45 68 88 95
FAX: 33-(0)1 40 61 30 74
email: ale...@pasteur.fr





Disclaimer
This communication is confidential and may contain privileged 
information intended solely for the named addressee(s). It may not be 
used or disclosed except for the purpose for which it has been sent. 
If you are not the intended recipient you must not review, use, 
disclose, copy, distribute or take any action in reliance upon it. If 
you have received this communication in error, please notify Astex 
Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and 
destroy all copies of the message and any attached documents.
Astex Therapeutics Ltd monitors, controls and protects all its 
messaging traffic in compliance with its corporate email policy. The 
Company accepts no liability or responsibility for any onward 
transmission or use of emails and attachments havin

Re: [ccp4bb] TLS refinement in Refmac gets stuck

2009-02-24 Thread Garib Murshudov 
Child killing and/or wrong bus seems to be related with static  
compilation on Mac intel OS 10.5. Existence of this bug is the reason  
why the version with bug fixes was not announced.

If you build from source code then this bug can be overcomed
Download source files and copy them to ccp4 source file directory and  
build from source.

Source codes can be found www.ysbl.york.ac.uk/refmac/latest_refmac.html
After gunzipping and untarring (it will work if you have build ccp4  
from source)


cp *.f $CPROG/refmac5_/
cp *.fh $CPROG/refmac5_/
cp *.c $CPROG/refmac5_/
cp *.cpp $CPROG/refmac5_/
cp *.h $CPROG/refmac5_/
cd $CPROG
rm *.o
make refmac5
cp refmac5 $CBIN/


After this procedure bus should be fine and child will also be safe.

Garib

On 24 Feb 2009, at 14:16, Anita Lewit-Bentley wrote:


Hi Ian,

A bug was reported with that version of Refmac, though I've no idea  
if that would cause your problem: you should upgrade to the latest  
version from the York website.


Done - and here is what happens:

1) the latest CCP4 package (6.1.1) runs the same version of Refmac  
(5.5.0066) and, predictably, crashes the same way;
2) on the Refmac webside there are much more recent versions of  
Refmac, not yet incorporated into the CCP4 package. Versions 72 and  
higher have a bug fix for TLS refinement.
3) included version 5.5.0086 (the precompiled version for Mac -  
intel) in CCP4  6.1 - and that runs fine 


except that it exits with a "bus error, child killed"

I HATE seeing a child killed 

but anyway, the output pdb and mtz files exist in the "scratch"  
directory with a .tmp extension, but otherwise perfectly useable.


So I can scrape along if I remember to copy the .tmp files accross  
to my working directory. Better than nothing!


Thanks for your hint.

Cheers,

Anita




-Original Message-
From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk 
] On

Behalf Of Anita Lewit-Bentley
Sent: 23 February 2009 15:31
To: CCP4BB@JISCMAIL.AC.UK
Subject: TLS refinement in Refmac gets stuck

Dear ccp4 folks,

I am refining a structure in several crystal forms, with several  
molecules
in the asymmetric unit. The structure is rather flexible, so I am  
using
TLS refinement, having first submitted the coordinates to the  
TLSMD server

(http://skuld.bmsc.washington.edu/~tlsmd/
 ).

The TLS refinement worked OK with REFMAC 5.2.0019 (CCP4 6.0.2) for 3
molecules in the asu, but not for crystal forms with 6 molecules/ 
asu (it

just got hung).

For the latter crystal forms I have switched to CCP4 6.1.0 with  
REFMAC
5.5.0066, where it worked fine for two crystals - and now plays up  
for the

third crystal!

It is not exactly crashing, so I don't know what is going on. This  
is the

last part of the logfile:

"Problem
xyz 1230   5.6147661  -5.1691742   2.8234024 -
1.55429840E-02  9.35771316E-02  0.17803398  0.23723726  
-0.13626081
5.39243035E-02 -0.14011805 -1.55429840E-02  0.11864197   
0.40574944

0.71992165  0.57164854 -0.39361250
**
*
* Information from CCP4Interface script
**
*
Writing final coordinates (XYZOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.pdb
**
*

**
*
* Information from CCP4Interface script
**
*
Writing final phases (HKLOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3M_Se_TLS_refmac1.mtz
**
*

**
*
* Information from CCP4Interface script
**
*
Writing final TLS (TLSOUT) to
/Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.tls
**
*
"
Unfortunately, there are NO files written out! There is no real  
error
message either, the CCP4i interface says the job has finished  
normally.


I have already tried to vary the number of TLS fragments, with no  
better

result. The older Refmac just gets hung

Any help will be appreciated!

Thanks,

Anita


Anita Lewit-Bentley

Unité d'Immunologie Structurale
CNRS URA 2185
Département de Biologie Structurale & Chimie
Institut Pasteur
25 rue du Dr. Roux
75724 Paris cedex 15
FRANCE

Tel: 33- (0)1 45 68 88 95
FAX: 33-(0)1 40 61 30 74
email: ale...@pasteur.fr





Disclaimer
This communication is confidential and may contain privileged  
information intended solely for the named addressee(s). It may not  
be used or disclosed except for

Re: [ccp4bb] TLS refinement in Refmac gets stuck

2009-02-24 Thread Graeme Winter 
Hi Folks,

If anyone wants to do this on an Intel mac you will need a fortran
compiler, which is not installed by XCode. There is a compatible
gfortran here:

http://r.research.att.com/tools/

which in my experience works fine, though is completely incapable of
producing static binaries.

There are instructions for the source-code build here:

http://www.ccp4wiki.org/~ccp4wiki/wiki/index.php?title=CCP4_6.1_installation#Source_Code_Installation

and here:

http://www.ccp4wiki.org/~ccp4wiki/wiki/index.php?title=CCP4_installation#Source_Code_Installation

Unfortunately, because the CCP4 release is built with the Intel
compilers (not gfortran) for this platform you can't just drop refmac5
onto an existing CCP4 installation and build, so you have to build
everything :o(

Garib: what compilers do you use such that someone building it
themselves will get a version which works, but using your same source
code and your compilers it does not?

Best wishes,

Graeme

2009/2/24 Garib Murshudov :
> Child killing and/or wrong bus seems to be related with static compilation
> on Mac intel OS 10.5. Existence of this bug is the reason why the version
> with bug fixes was not announced.
> If you build from source code then this bug can be overcomed
> Download source files and copy them to ccp4 source file directory and build
> from source.
> Source codes can be found www.ysbl.york.ac.uk/refmac/latest_refmac.html
> After gunzipping and untarring (it will work if you have build ccp4 from
> source)
>
> cp *.f $CPROG/refmac5_/
> cp *.fh $CPROG/refmac5_/
> cp *.c $CPROG/refmac5_/
> cp *.cpp $CPROG/refmac5_/
> cp *.h $CPROG/refmac5_/
> cd $CPROG
> rm *.o
> make refmac5
> cp refmac5 $CBIN/
>
>
> After this procedure bus should be fine and child will also be safe.
>
> Garib
>
> On 24 Feb 2009, at 14:16, Anita Lewit-Bentley wrote:
>
>> Hi Ian,
>>
>>> A bug was reported with that version of Refmac, though I've no idea if
>>> that would cause your problem: you should upgrade to the latest version from
>>> the York website.
>>
>> Done - and here is what happens:
>>
>> 1) the latest CCP4 package (6.1.1) runs the same version of Refmac
>> (5.5.0066) and, predictably, crashes the same way;
>> 2) on the Refmac webside there are much more recent versions of Refmac,
>> not yet incorporated into the CCP4 package. Versions 72 and higher have a
>> bug fix for TLS refinement.
>> 3) included version 5.5.0086 (the precompiled version for Mac - intel) in
>> CCP4  6.1 - and that runs fine 
>>
>> except that it exits with a "bus error, child killed"
>>
>> I HATE seeing a child killed 
>>
>> but anyway, the output pdb and mtz files exist in the "scratch" directory
>> with a .tmp extension, but otherwise perfectly useable.
>>
>> So I can scrape along if I remember to copy the .tmp files accross to my
>> working directory. Better than nothing!
>>
>> Thanks for your hint.
>>
>> Cheers,
>>
>> Anita
>>
>>>
 -Original Message-
 From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk]
 On
 Behalf Of Anita Lewit-Bentley
 Sent: 23 February 2009 15:31
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: TLS refinement in Refmac gets stuck

 Dear ccp4 folks,

 I am refining a structure in several crystal forms, with several
 molecules
 in the asymmetric unit. The structure is rather flexible, so I am using
 TLS refinement, having first submitted the coordinates to the TLSMD
 server
 (http://skuld.bmsc.washington.edu/~tlsmd/

  ).

 The TLS refinement worked OK with REFMAC 5.2.0019 (CCP4 6.0.2) for 3
 molecules in the asu, but not for crystal forms with 6 molecules/asu (it
 just got hung).

 For the latter crystal forms I have switched to CCP4 6.1.0 with REFMAC
 5.5.0066, where it worked fine for two crystals - and now plays up for
 the
 third crystal!

 It is not exactly crashing, so I don't know what is going on. This is
 the
 last part of the logfile:

 "Problem
 xyz         1230   5.6147661      -5.1691742       2.8234024     -
 1.55429840E-02  9.35771316E-02  0.17803398      0.23723726
 -0.13626081
 5.39243035E-02 -0.14011805     -1.55429840E-02  0.11864197
  0.40574944
 0.71992165      0.57164854     -0.39361250

 **
 *
 * Information from CCP4Interface script

 **
 *
 Writing final coordinates (XYZOUT) to
 /Users/anita/work/Integ/SOLEIL_09_08/3MSe_NTLS9_8_6_refmac1.pdb

 **
 *


 **
 *
 * Information from CCP4Interface script

[ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Mo Wong 
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!

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread artem 
Hi,

The question you have to ask yourself first is - does my gene actually
*have* the rare codons that you're trying to avoid? Experience shows that
usually it's not single codons that are a problem but pairs or triplets of
rare ones. If your gene does not have obviously bad codon combinations you
still may derive a benefit from codon optimization and especially from
mRNA structure shuffling. There are articles out there that attempt to
present statistically significant evidence and I tend to agree with them -
the practice of optimizing codon usage AND mRNA structure is a good and
useful one.

>From what you describe, you're working with a kinase. It is not at all
uncommon to have toxicity of kinases for E. coli. Additional issues
include heterogenous phosphorylation if the kinase is normally active or
can auto-activate in E.coli (classical example is PKA which can
phosphorylate itself in up to 30 places given the right conditions).

Toxicity isn't difficult to spot - classical signs include microcolonies,
heterogenous colonies, slow growth, plasmid instability and so on. Even
with a native gene you would likely notice these symptoms to some extent.

Incidentally - toxicity usually equals folding, i.e. for a kinase to be
toxic at least some of it has to be folded enough to work. This is
actually *good news* because toxicity can be combated on a different level
than lack of folded expression. For example, co-expression with
phosphatases tends to work miracles for kinase-based toxicity.

Finally, to answer your question directly - yes, i've seen several cases
of proteins not expressing even with rare codon tRNA supplemented in
trans, but expressing well from optimized DNA. Again - optimized for
codons AND structure, as I've never separated the two processes.

Synthetic DNA is cheap these days. If you can afford it - it's useful to
try before taking the next step. In this case the obvious next step is
attempt at expression in insect cells - kinases usually work out really
well in IC.

Artem

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Mo Wong 
Thanks for the reply.

I've checked my sequence for rare codons; however, what would be useful to a
pseudo-molecular biologist like me is a web server which will look at your
input DNA sequence and guesstimate the success of expression in E. coli
(i.e., consider codon frequency). Does one exist? Even better, a web server
that offers the choice of different commercial forms of E. coli (i.e.,
Rosetta and Codon+)? I tried Googling this a few days ago and didn't find
anything too useful?

Cheers!

On Tue, Feb 24, 2009 at 11:00 AM,  wrote:

> Hi,
>
> The question you have to ask yourself first is - does my gene actually
> *have* the rare codons that you're trying to avoid? Experience shows that
> usually it's not single codons that are a problem but pairs or triplets of
> rare ones. If your gene does not have obviously bad codon combinations you
> still may derive a benefit from codon optimization and especially from
> mRNA structure shuffling. There are articles out there that attempt to
> present statistically significant evidence and I tend to agree with them -
> the practice of optimizing codon usage AND mRNA structure is a good and
> useful one.
>
> From what you describe, you're working with a kinase. It is not at all
> uncommon to have toxicity of kinases for E. coli. Additional issues
> include heterogenous phosphorylation if the kinase is normally active or
> can auto-activate in E.coli (classical example is PKA which can
> phosphorylate itself in up to 30 places given the right conditions).
>
> Toxicity isn't difficult to spot - classical signs include microcolonies,
> heterogenous colonies, slow growth, plasmid instability and so on. Even
> with a native gene you would likely notice these symptoms to some extent.
>
> Incidentally - toxicity usually equals folding, i.e. for a kinase to be
> toxic at least some of it has to be folded enough to work. This is
> actually *good news* because toxicity can be combated on a different level
> than lack of folded expression. For example, co-expression with
> phosphatases tends to work miracles for kinase-based toxicity.
>
> Finally, to answer your question directly - yes, i've seen several cases
> of proteins not expressing even with rare codon tRNA supplemented in
> trans, but expressing well from optimized DNA. Again - optimized for
> codons AND structure, as I've never separated the two processes.
>
> Synthetic DNA is cheap these days. If you can afford it - it's useful to
> try before taking the next step. In this case the obvious next step is
> attempt at expression in insect cells - kinases usually work out really
> well in IC.
>
> Artem
>

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Partha Chakrabarti 
Just to add couple of things to what Artem said..

If it is something similar to a mammalian kinase or malaria protein for
example,

1. Recodonizaton can change expression from near zero to substantial
amounts, however,

a) ideally, it needs to be recodonized separately for each target expression
system (E. coli / yeast / IC)

b) Expression does not guaranty solubility.

One thing which sometimes works is coexpression of a phosphatase, if the non
phospho form is of any interest. Similar things have been done by Src
related kinases.. YopH or Lambda might be a good starting point. See:

http://www.ncbi.nlm.nih.gov/pubmed/16260764?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

HTH, Partha



On Tue, Feb 24, 2009 at 3:48 PM, 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!
>



-- 
MRC National Institute for Medical Research
Division of Molecular Structure
The Ridgeway, NW7 1AA, UK
Email: pcha...@nimr.mrc.ac.uk
Phone: + 44 208 816 2515

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Raphael Gasper 

This website is quite useful (via Expasy):

http://gcua.schoedl.de/

Raphael



Mo Wong schrieb:

Thanks for the reply.

I've checked my sequence for rare codons; however, what would be 
useful to a pseudo-molecular biologist like me is a web server which 
will look at your input DNA sequence and guesstimate the success of 
expression in E. coli (i.e., consider codon frequency). Does one 
exist? Even better, a web server that offers the choice of different 
commercial forms of E. coli (i.e., Rosetta and Codon+)? I tried 
Googling this a few days ago and didn't find anything too useful?


Cheers!

On Tue, Feb 24, 2009 at 11:00 AM, > wrote:


Hi,

The question you have to ask yourself first is - does my gene actually
*have* the rare codons that you're trying to avoid? Experience
shows that
usually it's not single codons that are a problem but pairs or
triplets of
rare ones. If your gene does not have obviously bad codon
combinations you
still may derive a benefit from codon optimization and especially from
mRNA structure shuffling. There are articles out there that attempt to
present statistically significant evidence and I tend to agree
with them -
the practice of optimizing codon usage AND mRNA structure is a
good and
useful one.

>From what you describe, you're working with a kinase. It is not
at all
uncommon to have toxicity of kinases for E. coli. Additional issues
include heterogenous phosphorylation if the kinase is normally
active or
can auto-activate in E.coli (classical example is PKA which can
phosphorylate itself in up to 30 places given the right conditions).

Toxicity isn't difficult to spot - classical signs include
microcolonies,
heterogenous colonies, slow growth, plasmid instability and so on.
Even
with a native gene you would likely notice these symptoms to some
extent.

Incidentally - toxicity usually equals folding, i.e. for a kinase
to be
toxic at least some of it has to be folded enough to work. This is
actually *good news* because toxicity can be combated on a
different level
than lack of folded expression. For example, co-expression with
phosphatases tends to work miracles for kinase-based toxicity.

Finally, to answer your question directly - yes, i've seen several
cases
of proteins not expressing even with rare codon tRNA supplemented in
trans, but expressing well from optimized DNA. Again - optimized for
codons AND structure, as I've never separated the two processes.

Synthetic DNA is cheap these days. If you can afford it - it's
useful to
try before taking the next step. In this case the obvious next step is
attempt at expression in insect cells - kinases usually work out
really
well in IC.

Artem




--
Raphael Gasper-Schönenbrücher
Max-Planck-Institut für molekulare Physiologie
Abt. für strukturelle Biologie
Otto-Hahn-Straße 11
44227 Dortmund, Germany
Tel: +49/231/133-2121
Fax: +49/231/133-2199

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Pascal Egea 
Hi Mo,
   Gene synthesis is definitely something you should try if you can afford
it.
However, I would suggest also trying to change expression plasmid and in
particular induction system and promoter system.
For toxic proteins we had some success using the pBAD (invitrogen)
expression system using arabinose induction and the arabinose operon
promoter different from the classical T7 promoter. It is tightly regulated
and has a very fast kinetic of induction. It is worth trying even combined
with gene synthesis.
   I also remember that some kinases are efficiently expressed in Ecoli in
presence of another expression plasmid encoding two " chaperones ", the
trigger factor and GroES/EL. This is worth trying too.

Hope this helps, cheers.

Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics
Stroud Lab

[ccp4bb] SCALA failed message

2009-02-24 Thread James Foadi 
Dear all,
I am running SCALA using ccp4i. The generated command file is:





***
/tmp/james/NONISO_insulin_from_liz_6_2_com.tmp
***
 title Scale merged insulin_from_liz _ Datasets in1, in2, in3, in4, in5
run 1 -
INCLUDE batch 1 to 100
run 2 -
INCLUDE batch 201 to 380
run 3 -
INCLUDE batch 401 to 522
run 4 -
INCLUDE batch 601 to 651
run 5 -
INCLUDE batch 701 to 720
RUN 2 reference
name run 1 project New crystal New dataset New
name run 2 project New crystal New dataset New
name run 3 project New crystal New dataset New
name run 4 project New crystal New dataset New
name run 5 project New crystal New dataset New
exclude EMAX -
10.0
partials -
check -
test 0.95 1.05 -
nogap
intensities INTEGRATED -
PARTIALS
final PARTIALS
scales -
rotation SPACING 5 -
secondary 6 -
bfactor ON -
BROTATION SPACING 20
UNFIX V
FIX A0
UNFIX A1
initial MEAN
tie surface 0.001
tie bfactor 0.3
cycles 10 converge 0.3 reject 2
output AVERAGE
print brief nooverlap
RSIZE 80
## This script run with the command   ##
# /home/james/build/ccp4-6.1.0/bin/scala HKLIN 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/exp_01/in1_in2_in3_in4_in5_MS_1_001.mtz"
 HKLOUT "/tmp/james/NONISO_insulin_from_liz_6_1_mtz.tmp" SCALES 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6.scala"
 ROGUES 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_rogues.log"
 NORMPLOT 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_normplot.xmgr"
 ANOMPLOT 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_anomplot.xmgr"
 PLOT 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_surface_plot.plt"
 CORRELPLOT 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_correlplot.xmgr"
 ROGUEPLOT
 
"/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_rogueplot.xmgr"



Unfortunately SCALA stops immediately with the following error message:


-
 ** Unrecognized keyword **

  RUN command must come before SCALES definition 

 ** Missing or illegal value **
-

Any idea on what's wrong? I have to say, I had run this before with similar 
data (up to 4 runs), and had never this problem.

Thank you!

J

 Dr James Foadi PhD
Membrane Protein Laboratory
Diamond Light Source Ltd.
Diamond House
Harwell Science and Innovation Campus
Didcot
Oxfordshire
OX11 0DE
United Kingdom


office email: james.fo...@diamond.ac.uk
alternative email: j.fo...@imperial.ac.uk
web page:

Re: [ccp4bb] SCALA failed message

2009-02-24 Thread Graeme Winter 
Hi James,

You are the victim of an epic command line. This has been fixed in the
6.1.1 release, but you will find that using a shorter path (i.e.
noniso rather than non-isomorphism &c.) will pull the command line
down to something more sensible.

CCP4i writes this out to allow the job to be rerun in a comment, but
the comment is too long for Scala!

Cheers,

Graeme

2009/2/24 James Foadi :
> Dear all,
> I am running SCALA using ccp4i. The generated command file is:
>
>
> 
>
>
> ***
> /tmp/james/NONISO_insulin_from_liz_6_2_com.tmp
> ***
>  title Scale merged insulin_from_liz _ Datasets in1, in2, in3, in4, in5
> run 1 -
>    INCLUDE batch 1 to 100
> run 2 -
>    INCLUDE batch 201 to 380
> run 3 -
>    INCLUDE batch 401 to 522
> run 4 -
>    INCLUDE batch 601 to 651
> run 5 -
>    INCLUDE batch 701 to 720
> RUN 2 reference
> name run 1 project New crystal New dataset New
> name run 2 project New crystal New dataset New
> name run 3 project New crystal New dataset New
> name run 4 project New crystal New dataset New
> name run 5 project New crystal New dataset New
> exclude EMAX -
>    10.0
> partials -
>    check -
>    test 0.95 1.05 -
>    nogap
> intensities INTEGRATED -
>    PARTIALS
> final PARTIALS
> scales -
>    rotation SPACING 5 -
>    secondary 6 -
>    bfactor ON -
>    BROTATION SPACING 20
> UNFIX V
> FIX A0
> UNFIX A1
> initial MEAN
> tie surface 0.001
> tie bfactor 0.3
> cycles 10 converge 0.3 reject 2
> output AVERAGE
> print brief nooverlap
> RSIZE 80
> ## This script run with the command   ##
> # /home/james/build/ccp4-6.1.0/bin/scala HKLIN 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/exp_01/in1_in2_in3_in4_in5_MS_1_001.mtz"
>  HKLOUT "/tmp/james/NONISO_insulin_from_liz_6_1_mtz.tmp" SCALES 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6.scala"
>  ROGUES 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_rogues.log"
>  NORMPLOT 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_normplot.xmgr"
>  ANOMPLOT 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_anomplot.xmgr"
>  PLOT 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_surface_plot.plt"
>  CORRELPLOT 
> "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_correlplot.xmgr"
>  ROGUEPLOT
>  "/home/james/gwyndaf/non-isomorphism/data_created/insulin_from_liz/NONISO_insulin_from_liz_6_rogueplot.xmgr"
> 
> 
>
> Unfortunately SCALA stops immediately with the following error message:
>
>
> -
>  ** Unrecognized keyword **
>
>   RUN command must come before SCALES definition 
>
>  ** Missing or illegal value **
> -
>
> Any idea on what's wrong? I have to say, I had run this before with similar 
> data (up to 4 runs), and had never this problem.
>
> Thank you!
>
> J
>
>  Dr James Foadi PhD
> Membrane Protein Laboratory
> Diamond Light Source Ltd.
> Diamond House
> Harwell Science and Innovation Campus
> Didcot
> Oxfordshire
> OX11 0DE
> United Kingdom
>
>
> office email: james.fo...@diamond.ac.uk
> alternative email: j.fo...@imperial.ac.uk
> web page:
>
>
>
>
>

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread artem 
Hi,

Servers to check for rare codons definitely do exist.

http://genomes.urv.es/OPTIMIZER/
http://www.doe-mbi.ucla.edu/~sumchan/caltor.html

and several others

As to comparing Rosetta and Codon+ - your best bet here is just to dig up
product literature from the manufacturers.

Rosetta uses pRARE: AGG, AGA, AUA, CUA, CCC, GGA
Codonplus
BL21-CodonPlus(DE3)-RIPL strain argU (AGA, AGG), ileY (AUA) , proL (CCC),
leuW (CUA)
BL21-CodonPlus-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA)
BL21-CodonPlus(DE3)-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA)
BL21-CodonPlus(DE3)-RIL-X strain argU (AGA, AGG), ileY (AUA), leuW (CUA)
BL21-CodonPlus-RP strain argU (AGA, AGG), proL (CCC)
BL21-CodonPlus(DE3)-RP strain argU (AGA, AGG), proL (CCC)
BL21-CodonPlus(DE3)-RP-X strain argU (AGA, AGG), proL (CCC)


Artem

> Thanks for the reply.
>
> I've checked my sequence for rare codons; however, what would be useful to
> a
> pseudo-molecular biologist like me is a web server which will look at your
> input DNA sequence and guesstimate the success of expression in E. coli
> (i.e., consider codon frequency). Does one exist? Even better, a web
> server
> that offers the choice of different commercial forms of E. coli (i.e.,
> Rosetta and Codon+)? I tried Googling this a few days ago and didn't find
> anything too useful?
>
> Cheers!
>
> On Tue, Feb 24, 2009 at 11:00 AM,  wrote:
>
>> Hi,
>>
>> The question you have to ask yourself first is - does my gene actually
>> *have* the rare codons that you're trying to avoid? Experience shows
>> that
>> usually it's not single codons that are a problem but pairs or triplets
>> of
>> rare ones. If your gene does not have obviously bad codon combinations
>> you
>> still may derive a benefit from codon optimization and especially from
>> mRNA structure shuffling. There are articles out there that attempt to
>> present statistically significant evidence and I tend to agree with them
>> -
>> the practice of optimizing codon usage AND mRNA structure is a good and
>> useful one.
>>
>> From what you describe, you're working with a kinase. It is not at all
>> uncommon to have toxicity of kinases for E. coli. Additional issues
>> include heterogenous phosphorylation if the kinase is normally active or
>> can auto-activate in E.coli (classical example is PKA which can
>> phosphorylate itself in up to 30 places given the right conditions).
>>
>> Toxicity isn't difficult to spot - classical signs include
>> microcolonies,
>> heterogenous colonies, slow growth, plasmid instability and so on. Even
>> with a native gene you would likely notice these symptoms to some
>> extent.
>>
>> Incidentally - toxicity usually equals folding, i.e. for a kinase to be
>> toxic at least some of it has to be folded enough to work. This is
>> actually *good news* because toxicity can be combated on a different
>> level
>> than lack of folded expression. For example, co-expression with
>> phosphatases tends to work miracles for kinase-based toxicity.
>>
>> Finally, to answer your question directly - yes, i've seen several cases
>> of proteins not expressing even with rare codon tRNA supplemented in
>> trans, but expressing well from optimized DNA. Again - optimized for
>> codons AND structure, as I've never separated the two processes.
>>
>> Synthetic DNA is cheap these days. If you can afford it - it's useful to
>> try before taking the next step. In this case the obvious next step is
>> attempt at expression in insect cells - kinases usually work out really
>> well in IC.
>>
>> Artem
>>
>

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Stephen Weeks 

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!

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread atul kumar 

hi
i am working on crystallisation of a 42 kDa his tag protein but,my protein gets 
precipitated at higher concenrtration till now i have not tried any salt to 
stabilise the protein,i am just confused what to do???should i go for higher 
salt concentration or anything alse.

-Original Message-
From: CCP4 bulletin board on behalf of Raphael Gasper
Sent: Tue 2/24/2009 9:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
 
This website is quite useful (via Expasy):

http://gcua.schoedl.de/

Raphael



Mo Wong schrieb:
> Thanks for the reply.
>
> I've checked my sequence for rare codons; however, what would be 
> useful to a pseudo-molecular biologist like me is a web server which 
> will look at your input DNA sequence and guesstimate the success of 
> expression in E. coli (i.e., consider codon frequency). Does one 
> exist? Even better, a web server that offers the choice of different 
> commercial forms of E. coli (i.e., Rosetta and Codon+)? I tried 
> Googling this a few days ago and didn't find anything too useful?
>
> Cheers!
>
> On Tue, Feb 24, 2009 at 11:00 AM,  > wrote:
>
> Hi,
>
> The question you have to ask yourself first is - does my gene actually
> *have* the rare codons that you're trying to avoid? Experience
> shows that
> usually it's not single codons that are a problem but pairs or
> triplets of
> rare ones. If your gene does not have obviously bad codon
> combinations you
> still may derive a benefit from codon optimization and especially from
> mRNA structure shuffling. There are articles out there that attempt to
> present statistically significant evidence and I tend to agree
> with them -
> the practice of optimizing codon usage AND mRNA structure is a
> good and
> useful one.
>
> >From what you describe, you're working with a kinase. It is not
> at all
> uncommon to have toxicity of kinases for E. coli. Additional issues
> include heterogenous phosphorylation if the kinase is normally
> active or
> can auto-activate in E.coli (classical example is PKA which can
> phosphorylate itself in up to 30 places given the right conditions).
>
> Toxicity isn't difficult to spot - classical signs include
> microcolonies,
> heterogenous colonies, slow growth, plasmid instability and so on.
> Even
> with a native gene you would likely notice these symptoms to some
> extent.
>
> Incidentally - toxicity usually equals folding, i.e. for a kinase
> to be
> toxic at least some of it has to be folded enough to work. This is
> actually *good news* because toxicity can be combated on a
> different level
> than lack of folded expression. For example, co-expression with
> phosphatases tends to work miracles for kinase-based toxicity.
>
> Finally, to answer your question directly - yes, i've seen several
> cases
> of proteins not expressing even with rare codon tRNA supplemented in
> trans, but expressing well from optimized DNA. Again - optimized for
> codons AND structure, as I've never separated the two processes.
>
> Synthetic DNA is cheap these days. If you can afford it - it's
> useful to
> try before taking the next step. In this case the obvious next step is
> attempt at expression in insect cells - kinases usually work out
> really
> well in IC.
>
> Artem
>
>

-- 
Raphael Gasper-Schönenbrücher
Max-Planck-Institut für molekulare Physiologie
Abt. für strukturelle Biologie
Otto-Hahn-Straße 11
44227 Dortmund, Germany
Tel: +49/231/133-2121
Fax: +49/231/133-2199