Re: [ccp4bb] Accidental crystallization of E. coli protein

[Bert Van-Den-Berg]

I do think one has to consider whether there is a sufficient scientific advance 
to justify publication. After all, peer reviewers are already overworked. From 
the information given I don't think I would try to publish this but I'd 
certainly consider depositing in the PDB and contaminant database. What about 
putting it in BioRxiv?


Bert



From: CCP4 bulletin board  on behalf of Debanu 

Sent: 05 April 2017 16:23
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Accidental crystallization of E. coli protein

I think it is worth reporting/publishing this crystallization and
structure in addition to depositing it in the PDB and
ContaMiner/ContaBase databases.

Items you can consider in reporting it based on the following but can
take a better decision if you complete the structure to deposition
quality:

a) Crystallization conditions are likely different from the
crystallized version from the recombinant expression?

b) Try to see why the resolution might be higher: crystal size,
crystallization conditions, x-ray source, processing and refinement
programs/current protocols? Always nice to have a higher resolution
and/or better refined current structure for others to use especially
if the target also has other potential importance. You never know when
someone later can use it for homology modeling or virtual library
screening.

c) As you complete full structure determination, you may also find
additional features in the maps/structure compared to previous
reports, i.e., differences around
functional sites possibly conformational variations, endogenous or
known densities that may have some functional implications, etc.

I also think publicly-funded research that involves a complete piece
of work that may be useful for others now or in the future should be
reported since publications are also record keeping for the future.
Everything does not have to have high scientific impact today.

Best,
Debanu
--
Debanu Das

On Wed, Apr 5, 2017 at 4:46 AM, Stefan Arold  wrote:
> Hi Mohamed,
> I don't recall such a case of a crystallized proteolytically cleaved
> contaminant (see our recent summary of contamination cases
> http://scripts.iucr.org/cgi-bin/paper?ei5009 , and references therein).
> I would certainly recommend depositing the data at the PDB - it may help
> others address questions that need the higher resolution, or facilitate MR.
> We can then also include your protein into our ContaMiner & ContaBase
> webserver & database. https://strube.cbrc.kaust.edu.sa/contaminer/
ContaMiner - 
strube.cbrc.kaust.edu.sa
strube.cbrc.kaust.edu.sa
ContaMiner What is it ? ContaMiner is a rapid automated large-scale detection 
of contaminant crystals. Protein contaminants, from the expression host, 
purification ...


> Best wishes
> Stefan
>
> On 5 April 2017 at 00:16, Mohamed Noor  wrote:
>>
>> During the crystallization of a totally unrelated protein from a different
>> bacterium in E. coli, we managed to somehow crystallize an E. coli protein.
>> It turned out to be only the catalytic domain of an enzyme. Two previous
>> reports both used recombinant expression of this enzyme followed by limited
>> proteolysis in order to crystallize this domain.
>>
>> Has something like this been reported before? I know the stories of AcrB
>> etc., but I am looking specifically for a 'naturally proteolyzed'
>> crystallization.
>>
>> Looking at our structure and the previously published one, there is not
>> much difference, with an rmsd of about 0.3 A but our data resolution is a
>> bit higher. This is perhaps unsurprising as the unit cells are very similar
>> (in fact, I just did a quick RBR). Should we bother depositing and
>> publishing this observation?
>>
>> Thanks.
>> Mohamed
>
>

[ccp4bb] Structural Bioinformatics Training Workshop & Hackathon 2017 - Application of Big Data Technology and 3D Visualization

[Peter Rose]

Structural Bioinformatics Training Workshop & Hackathon 2017 - Application
of Big Data Technology and 3D Visualization



San Diego Supercomputer Center/University of California, San Diego, June 26
- 28, 2017



https://www.etouches.com/mmtf2017



This 3-day hands-on workshop introduces participants to the development of
fast and scalable structural bioinformatics methods using state-of-the-art
Big Data technologies and Web-GL 3D visualization. The first two days of
the workshop combine lectures, hands-on applications, and programming
sessions. On the third day participants apply the new technologies to their
own projects.



This workshop is held at the University of California, San Diego and hosted
by the Structural Bioinformatics Laboratory at SDSC in collaboration with
the RCSB Protein Data Bank.



Sponsorship

This workshop is sponsored by the NIH Big Data to Knowledge (BD2K)
initiative. Air travel and 4-day lodging will be provided for
non-commercial participants, including a limited number of international
participants. Apply now to secure your place in the workshop. Participants
will be selected based on the best fit to the program. Application
deadline: April 30, 2017.



Target Audience

The workshop is aimed at graduate students, postdocs, staff, faculty,
industrial researchers, and scientific software developers who develop
software for Structural Bioinformatics applications. Intermediate to
advanced programming skills in high-level languages are required (Java,
JavaScript, Python, C++).



For more details and application see: https://www.etouches.com/mmtf2017


-- 
Peter Rose, Ph.D.
Director, Structural Bioinformatics Laboratory
San Diego Supercomputer Center
UC San Diego
+1-858-822-5497

Re: [ccp4bb] Accidental crystallization of E. coli protein

[Debanu]

I think it is worth reporting/publishing this crystallization and
structure in addition to depositing it in the PDB and
ContaMiner/ContaBase databases.

Items you can consider in reporting it based on the following but can
take a better decision if you complete the structure to deposition
quality:

a) Crystallization conditions are likely different from the
crystallized version from the recombinant expression?

b) Try to see why the resolution might be higher: crystal size,
crystallization conditions, x-ray source, processing and refinement
programs/current protocols? Always nice to have a higher resolution
and/or better refined current structure for others to use especially
if the target also has other potential importance. You never know when
someone later can use it for homology modeling or virtual library
screening.

c) As you complete full structure determination, you may also find
additional features in the maps/structure compared to previous
reports, i.e., differences around
functional sites possibly conformational variations, endogenous or
known densities that may have some functional implications, etc.

I also think publicly-funded research that involves a complete piece
of work that may be useful for others now or in the future should be
reported since publications are also record keeping for the future.
Everything does not have to have high scientific impact today.

Best,
Debanu
--
Debanu Das

On Wed, Apr 5, 2017 at 4:46 AM, Stefan Arold  wrote:
> Hi Mohamed,
> I don't recall such a case of a crystallized proteolytically cleaved
> contaminant (see our recent summary of contamination cases
> http://scripts.iucr.org/cgi-bin/paper?ei5009 , and references therein).
> I would certainly recommend depositing the data at the PDB - it may help
> others address questions that need the higher resolution, or facilitate MR.
> We can then also include your protein into our ContaMiner & ContaBase
> webserver & database. https://strube.cbrc.kaust.edu.sa/contaminer/
> Best wishes
> Stefan
>
> On 5 April 2017 at 00:16, Mohamed Noor  wrote:
>>
>> During the crystallization of a totally unrelated protein from a different
>> bacterium in E. coli, we managed to somehow crystallize an E. coli protein.
>> It turned out to be only the catalytic domain of an enzyme. Two previous
>> reports both used recombinant expression of this enzyme followed by limited
>> proteolysis in order to crystallize this domain.
>>
>> Has something like this been reported before? I know the stories of AcrB
>> etc., but I am looking specifically for a 'naturally proteolyzed'
>> crystallization.
>>
>> Looking at our structure and the previously published one, there is not
>> much difference, with an rmsd of about 0.3 A but our data resolution is a
>> bit higher. This is perhaps unsurprising as the unit cells are very similar
>> (in fact, I just did a quick RBR). Should we bother depositing and
>> publishing this observation?
>>
>> Thanks.
>> Mohamed
>
>

[ccp4bb] Post-doc in membrane protein structural biology, Karolinska Institute.

[Joseph Brock]

Dear colleagues,

Please see the information below regarding an opening in our research group. 
Please share with anyone who might be interested.

Thanks!

-Joseph.



A two-year post-doctoral position is available at the Department of Medical 
Biochemistry and Biophysics (MBB), Karolinska Institutet, Stockholm. The 
project deals primarily with over-expression, purification, crystallisation and 
structure determination of human membrane proteins involved in the generation 
of lipid mediators of inflammation. The successful candidate will join a 
multi-disciplinary research team and the position represents an opportunity for 
a postdoctoral scientist to develop their expertise as well as broaden their 
experience. The major interests of the lab are in membrane protein structure 
and function. We combine structural techniques such as macromolecular 
crystallography with biophysical, biochemical and computational methods to 
investigate the structural basis of catalysis. The lab has all necessary 
equipment and access to facilities needed for the project, including a core 
facility for protein science. We have periodic access to beamlines at ESRF 
(France), Diamond (UK), BESSY and DESY (Germany).

Qualifications:
The candidate should hold a PhD conferred within the previous two years (or 
expect conferral in the near future), be motivated to independently solve 
problems and a have a strong background in biochemistry or structural biology. 
Experience with protein expression, purification and biochemical 
characterisation is required.  Prior experience in membrane protein 
biochemistry and computational skills such as a familiarity with the LINUX 
operating system will be added advantages.

The application should contain a single PDF document including:

*   Letter of intention stating the scientific interests, technical skills, 
and previous experience of the candidate.

*   CV including date and field of (expected) graduation and list of 
publications, with the contact information of at least two references.

This should be sent to joseph.br...@ki.se and jesper.haeggst...@ki.se.

The deadline for application is the 28th of April, 2016.




Joseph Brock | PhD
Division of Physiological Chemistry II
Department of Medical Biochemistry and Biophysics
Karolinska Institutet
Scheeles väg 2
SE-171 77 Stockholm, Sweden

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

ah! ok, that's a different matter
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:29
An: Hughes, Jon ; CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

But I think he was asking about imaging of intrinsic fluorescence of protein 
crystals…

I like your idea about gel imaging, though.

JPK

From: Hughes, Jon [mailto:jon.hug...@bot3.bio.uni-giessen.de]
Sent: Wednesday, April 05, 2017 8:23 AM
To: Keller, Jacob >; 
CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] UVEX UV Fluorescence

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon 
>;
 CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

Re: [ccp4bb] UVEX UV Fluorescence

[Keller, Jacob]

But I think he was asking about imaging of intrinsic fluorescence of protein 
crystals…

I like your idea about gel imaging, though.

JPK

From: Hughes, Jon [mailto:jon.hug...@bot3.bio.uni-giessen.de]
Sent: Wednesday, April 05, 2017 8:23 AM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: AW: [ccp4bb] UVEX UV Fluorescence

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon 
>;
 CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

for ethidium bromide you need a 590nm (50-100nm FWHH) bandpass interference 
filter. ccd chips are sensitive to infrared and transilluminators produce a lot 
of it!
best
j

Von: Keller, Jacob [mailto:kell...@janelia.hhmi.org]
Gesendet: Mittwoch, 5. April 2017 14:16
An: Hughes, Jon ; CCP4BB@JISCMAIL.AC.UK
Betreff: RE: [ccp4bb] UVEX UV Fluorescence

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

Re: [ccp4bb] UVEX UV Fluorescence

[Keller, Jacob]

Why 590 nm? BP, SP, LP? I would have thought 390 nm LP or similar--was it a 
typo?

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Hughes, 
Jon
Sent: Wednesday, April 05, 2017 6:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

Re: [ccp4bb] Accidental crystallization of E. coli protein

[Stefan Arold]

Hi Mohamed,
I don't recall such a case of a crystallized proteolytically cleaved
contaminant (see our recent summary of contamination cases
http://scripts.iucr.org/cgi-bin/paper?ei5009 , and references therein).
I would certainly recommend depositing the data at the PDB - it may help
others address questions that need the higher resolution, or facilitate MR.
We can then also include your protein into our ContaMiner & ContaBase
webserver & database. https://strube.cbrc.kaust.edu.sa/contaminer/
Best wishes
Stefan

On 5 April 2017 at 00:16, Mohamed Noor  wrote:

> During the crystallization of a totally unrelated protein from a different
> bacterium in E. coli, we managed to somehow crystallize an E. coli protein.
> It turned out to be only the catalytic domain of an enzyme. Two previous
> reports both used recombinant expression of this enzyme followed by limited
> proteolysis in order to crystallize this domain.
>
> Has something like this been reported before? I know the stories of AcrB
> etc., but I am looking specifically for a 'naturally proteolyzed'
> crystallization.
>
> Looking at our structure and the previously published one, there is not
> much difference, with an rmsd of about 0.3 A but our data resolution is a
> bit higher. This is perhaps unsurprising as the unit cells are very similar
> (in fact, I just did a quick RBR). Should we bother depositing and
> publishing this observation?
>
> Thanks.
> Mohamed
>

[ccp4bb] AW: [ccp4bb] UVEX UV Fluorescence

[Hughes, Jon]

you can save a fortune by just fitting a 590nm interference filter to the front 
of a ccd camera attached to a PC + a UV/B transilluminator. i set this up in my 
lab for about €1000 total 15 years ago and we've used it everyday since.
cheers
jon

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Cyprian 
Cukier
Gesendet: Mittwoch, 5. April 2017 12:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] UVEX UV Fluorescence

Dear All,

We are about to buy a new imaging system for our laboratories and we consider 
UVEX UV Fluorescence Imaging from MD. Can anyone provide some comments about 
this equipment (eg. is it reliable, robust, no technical issues, 
low-maintenance etc.)? All comments will be appreciated.

Thanks,
Cyprian

[ccp4bb] UVEX UV Fluorescence

[Cyprian Cukier]

Dear All,

 

We are about to buy a new imaging system for our laboratories and we
consider UVEX UV Fluorescence Imaging from MD. Can anyone provide some
comments about this equipment (eg. is it reliable, robust, no technical
issues, low-maintenance etc.)? All comments will be appreciated.

 

Thanks,

Cyprian

 

Re: [ccp4bb] Ambiguous crystal diffraction pattern

[Kelvin Lau]

Seen this before. Citrate crystals 


-- 
Kelvin Lau 
Structural Plant Biology Laboratory 
Department of Botany and Plant Biology 
Science III 
University of Geneva 
30 Quai E. Ansermet 
1211 Geneva 
Switzerland 
Email: kelvin@unige.ch 
Phone: +41 22 379 3026






On Tue, Apr 4, 2017 at 2:41 PM +0200, "Prashant Deshmukh" 
 wrote:










Dear Crystallographers,

Please help us in figuring out whether the attached crystal diffraction 
screenshot belongs to a protein crystal or that of a salt crystal. The 
cryoprotection condition was not optimized for the crystal. The crystallization 
conditions for the crystal is : 
Ammonium sulphate , lithium sulphate, Sodium Citrate buffer pH = 5.6. 

Thanks.Prashant DeshmukhDept. of Biophysics,NIMHANS,Bangalore 560 
029,E-mail:prashantbiophys...@gmail.com
Mob.No.: +919620986525

Re: [ccp4bb] Accidental crystallization of E. coli protein

[zaigham mahmood khan]

This may seem an interesting case, given that protocol fur purification,
and subsequent crystallization is reproducible as well as this enzyme is a
valid drug target for pathogenic strains of E. coli.

Best
Z
On Apr 5, 2017 02:51, "Vipul Panchal"  wrote:

> I don't think it is going to be any scientific story. Even if you think of
> publishing what aspect are you going to discuss? What new are you going to
> give to the community.
>
> On Wed, Apr 5, 2017 at 2:46 AM, Mohamed Noor  > wrote:
>
>> During the crystallization of a totally unrelated protein from a
>> different bacterium in E. coli, we managed to somehow crystallize an E.
>> coli protein. It turned out to be only the catalytic domain of an enzyme.
>> Two previous reports both used recombinant expression of this enzyme
>> followed by limited proteolysis in order to crystallize this domain.
>>
>> Has something like this been reported before? I know the stories of AcrB
>> etc., but I am looking specifically for a 'naturally proteolyzed'
>> crystallization.
>>
>> Looking at our structure and the previously published one, there is not
>> much difference, with an rmsd of about 0.3 A but our data resolution is a
>> bit higher. This is perhaps unsurprising as the unit cells are very similar
>> (in fact, I just did a quick RBR). Should we bother depositing and
>> publishing this observation?
>>
>> Thanks.
>> Mohamed
>>
>
>
>
> --
> Vipul Panchal
> Senior Research Fellow,
> Respiratory disease and biology,
> CSIR-IGIB
> (M)-9540113372
>

Re: [ccp4bb] Accidental crystallization of E. coli protein

[Vipul Panchal]

I don't think it is going to be any scientific story. Even if you think of
publishing what aspect are you going to discuss? What new are you going to
give to the community.

On Wed, Apr 5, 2017 at 2:46 AM, Mohamed Noor 
wrote:

> During the crystallization of a totally unrelated protein from a different
> bacterium in E. coli, we managed to somehow crystallize an E. coli protein.
> It turned out to be only the catalytic domain of an enzyme. Two previous
> reports both used recombinant expression of this enzyme followed by limited
> proteolysis in order to crystallize this domain.
>
> Has something like this been reported before? I know the stories of AcrB
> etc., but I am looking specifically for a 'naturally proteolyzed'
> crystallization.
>
> Looking at our structure and the previously published one, there is not
> much difference, with an rmsd of about 0.3 A but our data resolution is a
> bit higher. This is perhaps unsurprising as the unit cells are very similar
> (in fact, I just did a quick RBR). Should we bother depositing and
> publishing this observation?
>
> Thanks.
> Mohamed
>



-- 
Vipul Panchal
Senior Research Fellow,
Respiratory disease and biology,
CSIR-IGIB
(M)-9540113372

Re: [ccp4bb] Off-topic: Attach His-tagged Protein to Coverslip

[Tristan Croll]

One possibility: surface-immobilised forms of the peptide HGGHHG bind strongly 
to His tags (coordinating around zinc rather than nickel). See 
https://www.ncbi.nlm.nih.gov/m/pubmed/15050643/. If you want true permanence, 
you could apply the trick used on BiaCore chips. They use a NTA-functional 
carboxymethyldextran surface. His-tagged proteins are captured as usual, then 
rinsed and made permanent by incubating with some water-soluble carbodiimide to 
link the carboxymethyl groups to surface lysines - the advantage being that all 
the proteins are in essentially the same orientation. Not sure if anyone sells 
coverslip equivalents.

Best regards,

Tristan

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 5 Apr 2017, at 04:19, Keller, Jacob  wrote:
> 
> Does anyone have a simple way to attach purified his-tagged protein solidly 
> to a coverslip?
>  
> Thanks,
>  
> Jacob Keller
>  
> ***
> Jacob Pearson Keller, PhD
> Research Scientist
> HHMI Janelia Research Campus / Looger lab
> Phone: (571)209-4000 x3159
> Email: kell...@janelia.hhmi.org
> ***
>  

Re: [ccp4bb] FW: [ccp4bb] Using a codon-optimised gene to improve protein solubility

[AJV]

Hi Sutapa,

The input from other commenters has been great, so I'll stick to discussing 
only the portion of your question regarding baculovirus.

In our experience, codon-optimization has resulted in little to no improvement 
of total protein yields or protein solubility in baculovirus. We routinely work 
on challenging targets (mammalian membrane proteins), and while we don't have 
mounds of data on the subject, we have expressed enough eukaryotic protein 
families to say this with some confidence. Our workaround to bad expressers has 
been to screen orthologs of the protein of interest, using life's natural codon 
diversity to our advantage.

That being said, my suggestion would be to attempt baculovirus again, as there 
must have been a good reason for you to try it in the first place. Not knowing 
whether your gene is eukaryotic, specifically mammalian, or if 
post-translational modifications could be important; but if any of these are 
the case, baculovirus would be one of the best systems to pound on. 

We test several variables that always result in "seeing" some amount of protein 
expression with baculovirus (depending on detection method) -- time, 
temperature, and MOI. Once high titer / low passage virus is obtained and 
titered (can't stress the importance of titering enough), I usually set up six 
50mL suspension cultures of Sf9 cells and infect them at 2x10^6 cells/mL, using 
MOIs of 0.2, 2.0, and 20.0, in duplicate. After 24hrs at 27°C, I place three of 
the cultures at each MOI in an identical shaking incubator at 20-23°C, and 
leave the other three at 27°C. I then remove ~5mL of culture at various time 
points (about every 12 or 24hrs depending on scheduling), up to ~120hrs for all 
six samples. Then, crack the cells and use your detection method of choice 
(GFP, Western, Coomassie) to assess expression. This workflow can be repeated 
using other insect cell types (Sf21, Tn5), as they all will vary in their 
protein expression capabilities. 

Hope this answers part of your question, and results in some quality sample.

Best,

Alex
_
Alex J. Vecchio, Ph.D.
Postdoctoral Fellow
University of California, San Francisco
Department of Biochemistry & Biophysics
Laboratory of Robert M. Stroud, Ph.D.