[ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins

2022-02-19 Thread Casper Wilkens 
Is absorbance at 205 nm not a possibility, Jack?


Casper Wilkens
Asst. Prof.

Structural Enzymology & Biorefining
DTU Bioengineering




Technical University of Denmark
[http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif]

Department of Biotechnology and Biomedicine
Søltofts Plads
Building 224
Room 028

2800  Kgs. Lyngby
c...@dtu.dk
www.bioengineering.dtu.dk/
https://www.cazypedia.org/index.php/User:Casper_Wilkens
https://twitter.com/protein_artist
https://www.researchgate.net/profile/Casper_Wilkens





Fra: CCP4 bulletin board  på vegne af Artem Evdokimov 

Sendt: 19. februar 2022 02:12:50
Til: CCP4BB@JISCMAIL.AC.UK
Emne: Re: [ccp4bb] Strategies for increasing Trp content of proteins

We have had success placing single trp on the outside of proteins, as weird as 
it sounds it works pretty well. If your protein is easy to express you can try 
a few choices.

If your protein is difficult to express, a fusion with a trp-positive domain or 
with gfp may be a better option.

There are many other options but their application is best considered with more 
information in hand

Artem

On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:

Dear CCP4BB,

We are working on a protein that has no Trp residues, which makes 
chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% = 
0.104). Does anyone have experience using mutagenesis to increase the Trp 
content of proteins?

Thanks,

Jack Tanner

--
John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
Department of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Email: tanne...@missouri.edu
https://cafnrfaculty.missouri.edu/tannerlab/
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A






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Re: [ccp4bb] Strategies for increasing Trp content of proteins / Tris-NTA-Orange-Green

2022-02-19 Thread mesters 
Another possibility you might like to consider is to make use of 
non-covalent-binding dyes:


1) If your protein contains a His6-tag, you could add a bit of a (for 
partial non-covalent labeling) Tris–nitrilotriacetic acid 
(Tris-NTA)–conjugated based dye/fluorophore such as 
Tris-NTA-Orange-Green See https://doi.org/10.2144/000113551 



2) If no His6-tag, dependent on the protein nature (hydrophobic vs. 
charged/hydrophylic, etc.), you could add (again partial labelling) make 
use of cyanine or squarylium dyes... See https://doi.org/10.3390/90300040


- Jeroen -



Am 19.02.22 um 09:18 schrieb Casper Wilkens:


Is absorbance at 205 nm not a possibility, Jack?


Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering

Technical University of Denmark


Department of Biotechnology and Biomedicine
Søltofts Plads
Building 224
Room 028
2800  Kgs. Lyngby
c...@dtu.dk 
www.bioengineering.dtu.dk/ 
https://www.cazypedia.org/index.php/User:Casper_Wilkens 


https://twitter.com/protein_artist 
https://www.researchgate.net/profile/Casper_Wilkens 




*Fra:* CCP4 bulletin board  på vegne af Artem 
Evdokimov 

*Sendt:* 19. februar 2022 02:12:50
*Til:* CCP4BB@JISCMAIL.AC.UK
*Emne:* Re: [ccp4bb] Strategies for increasing Trp content of proteins
We have had success placing single trp on the outside of proteins, as 
weird as it sounds it works pretty well. If your protein is easy to 
express you can try a few choices.


If your protein is difficult to express, a fusion with a trp-positive 
domain or with gfp may be a better option.


There are many other options but their application is best considered 
with more information in hand


Artem

On Fri, Feb 18, 2022, 8:02 PM Tanner, John J.  
wrote:


Dear CCP4BB,

We are working on a protein that has no Trp residues, which makes
chromatography challenging due to the low absorbance at 280 nm
(Abs 0.1% = 0.104). Does anyone have experience using mutagenesis
to increase the Trp content of proteins?

Thanks,

Jack Tanner

-- 


John J. Tanner

Professor of Biochemistry and Chemistry

Associate Chair of Biochemistry

Department of Biochemistry

University of Missouri
117 Schweitzer Hall

503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280

Email: tanne...@missouri.edu 
https://cafnrfaculty.missouri.edu/tannerlab/


Lab: Schlundt Annex rooms 3,6,9, 203B, 203C

Office: Schlundt Annex 203A




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--
signature.html *Dr. /math. et dis. nat./ Jeroen R. Mesters
*Deputy, Lecturer, Program Coordinator Infection Biology 

Visiting Professorship (South Bohemian University 
) in Biophysics


*University of Lübeck*
Center for Structural and Cell Biology in Medicine*
Institute of Biochemistry*

Tel  +49 451 3101 3105 (Secretariate 3101)
Fax +49 451 3101 3104 *
*jeroen.mest...@uni-luebeck.de
https://www.biochem.uni-luebeck.de
https://orcid.org/-0001-8532-6699

*Ratzeburger Allee 160
23538 Lübeck, Schleswig-Holstein
Germany*



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Re: [ccp4bb] Strategies for increasing Trp content of proteins

2022-02-19 Thread Tanner, John J. 
Casper: You make a good point about using 205 nm. I checked our system this 
morning and the wavelength is fixed at 280 nm (U9-L).

Artem: Did you consider secondary structure and/or amino acid residue type when 
choosing sites for mutagenesis? Is your work published?


From: CCP4 bulletin board  on behalf of Casper Wilkens 
<5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>
Date: Saturday, February 19, 2022 at 2:19 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins
WARNING: This message has originated from an External Source. This may be a 
phishing expedition that can result in unauthorized access to our IT System. 
Please use proper judgment and caution when opening attachments, clicking 
links, or responding to this email.

Is absorbance at 205 nm not a possibility, Jack?


Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering


Technical University of Denmark
[http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif]
Department of Biotechnology and Biomedicine
Søltofts Plads
Building 224
Room 028
2800  Kgs. Lyngby
c...@dtu.dk
www.bioengineering.dtu.dk/
https://www.cazypedia.org/index.php/User:Casper_Wilkens
https://twitter.com/protein_artist
https://www.researchgate.net/profile/Casper_Wilkens

Fra: CCP4 bulletin board  på vegne af Artem Evdokimov 

Sendt: 19. februar 2022 02:12:50
Til: CCP4BB@JISCMAIL.AC.UK
Emne: Re: [ccp4bb] Strategies for increasing Trp content of proteins

We have had success placing single trp on the outside of proteins, as weird as 
it sounds it works pretty well. If your protein is easy to express you can try 
a few choices.

If your protein is difficult to express, a fusion with a trp-positive domain or 
with gfp may be a better option.

There are many other options but their application is best considered with more 
information in hand

Artem

On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:

Dear CCP4BB,

We are working on a protein that has no Trp residues, which makes 
chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% = 
0.104). Does anyone have experience using mutagenesis to increase the Trp 
content of proteins?

Thanks,

Jack Tanner

--
John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
Department of Biochemistry
University of Missouri
117 Schweitzer Hall
503 S College Avenue
Columbia, MO 65211
Phone: 573-884-1280
Email: tanne...@missouri.edu
https://cafnrfaculty.missouri.edu/tannerlab/
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A






To unsubscribe from the CCP4BB list, click the following link:
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To unsubsc

Re: [ccp4bb] Strategies for increasing Trp content of proteins

2022-02-19 Thread Weidenhausen, Jonas 
Dear Jack,

You may want to consider using a Strep2 tag (instead of your tag or additional 
to it) as it contains one Trp. I used it for some of my smaller proteins 
(without natural Trp), which works fine for detection and purification. 
Typically, I would go for N-terminal His and C-terminal Strep2, so you can be 
sure to purify homogenous FL proteins.

Cheers,
Jonas

--
Jonas Weidenhausen
PhD Student
AG Sinning

BZH Heidelberg University, room: 524
INF 328, 69120 Heidelberg
Phone: +49 6221 54-4786
jonas.weidenhau...@bzh.uni-heidelberg.de

On 19. Feb 2022, at 19:42, Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:

Casper: You make a good point about using 205 nm. I checked our system this 
morning and the wavelength is fixed at 280 nm (U9-L).

Artem: Did you consider secondary structure and/or amino acid residue type when 
choosing sites for mutagenesis? Is your work published?


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Casper Wilkens 
<5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>
Date: Saturday, February 19, 2022 at 2:19 AM
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins
WARNING: This message has originated from an External Source. This may be a 
phishing expedition that can result in unauthorized access to our IT System. 
Please use proper judgment and caution when opening attachments, clicking 
links, or responding to this email.

Is absorbance at 205 nm not a possibility, Jack?



Casper Wilkens

Asst. Prof.

Structural Enzymology & Biorefining

DTU Bioengineering




Technical University of Denmark


[http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif]

Department of Biotechnology and Biomedicine

Søltofts Plads
Building 224
Room 028

2800  Kgs. Lyngby

c...@dtu.dk

www.bioengineering.dtu.dk/

https://www.cazypedia.org/index.php/User:Casper_Wilkens
https://twitter.com/protein_artist
https://www.researchgate.net/profile/Casper_Wilkens



Fra: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
på vegne af Artem Evdokimov 
mailto:artem.evdoki...@gmail.com>>
Sendt: 19. februar 2022 02:12:50
Til: CCP4BB@JISCMAIL.AC.UK
Emne: Re: [ccp4bb] Strategies for increasing Trp content of proteins

We have had success placing single trp on the outside of proteins, as weird as 
it sounds it works pretty well. If your protein is easy to express you can try 
a few choices.

If your protein is difficult to express, a fusion with a trp-positive domain or 
with gfp may be a better option.

There are many other options but their application is best considered with more 
information in hand

Artem

On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:

Dear CCP4BB,

We are working on a protein that has no Trp residues, which makes 
chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% = 
0.104). Does anyone have experience using mutagenesis to increase the Trp 
content of proteins?

Thanks,

Jack Tanner

--
John J. Tanner
Professor of Biochemistry and Chemistry
Associate Chair of Biochemistry
Department of Bi

Re: [ccp4bb] Strategies for increasing Trp content of proteins

2022-02-19 Thread Artem Evdokimov 
Yes we chose the sites based on predicted or known structure, to be sure.
No that was not a published thing, curse of commercial work :(

Happy to help on a private channel.

Artem

On Sat, Feb 19, 2022, 1:42 PM Tanner, John J.  wrote:

> Casper: You make a good point about using 205 nm. I checked our system
> this morning and the wavelength is fixed at 280 nm (U9-L).
>
>
>
> Artem: Did you consider secondary structure and/or amino acid residue type
> when choosing sites for mutagenesis? Is your work published?
>
>
>
>
>
> *From: *CCP4 bulletin board  on behalf of Casper
> Wilkens <5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>
> *Date: *Saturday, February 19, 2022 at 2:19 AM
> *To: *CCP4BB@JISCMAIL.AC.UK 
> *Subject: *[ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of
> proteins
>
> *WARNING:* This message has originated from an External Source. This may
> be a phishing expedition that can result in unauthorized access to our IT
> System. Please use proper judgment and caution when opening attachments,
> clicking links, or responding to this email.
>
> Is absorbance at 205 nm not a possibility, Jack?
>
>
>
> *Casper Wilkens *
>
> Asst. Prof.
>
> Structural Enzymology & Biorefining
>
> DTU Bioengineering
>
>
>
> *Technical University of Denmark*
>
> Department of Biotechnology and Biomedicine
>
> Søltofts Plads
> Building 224
> Room 028
>
> 2800  Kgs. Lyngby
>
> c...@dtu.dk 
>
> www.bioengineering.dtu.dk/
> 
>
> https://www.cazypedia.org/index.php/User:Casper_Wilkens
> 
> https://twitter.com/protein_artist
> 
> https://www.researchgate.net/profile/Casper_Wilkens
> 
> --
>
> *Fra:* CCP4 bulletin board  på vegne af Artem
> Evdokimov 
> *Sendt:* 19. februar 2022 02:12:50
> *Til:* CCP4BB@JISCMAIL.AC.UK
> *Emne:* Re: [ccp4bb] Strategies for increasing Trp content of proteins
>
>
>
> We have had success placing single trp on the outside of proteins, as
> weird as it sounds it works pretty well. If your protein is easy to express
> you can try a few choices.
>
>
>
> If your protein is difficult to express, a fusion with a trp-positive
> domain or with gfp may be a better option.
>
>
>
> There are many other options but their application is best considered with
> more information in hand
>
>
>
> Artem
>
>
>
> On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. 
> wrote:
>
> Dear CCP4BB,
>
> We are working on a protein that has no Trp residues, which makes
> chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% =
> 0.104). Does anyone have experience using mutagenesis to increase the Trp
> content of proteins?
>
> Thanks,
>
> Jack Tanner
>
>
>
> --
>
> John J. Tanner
>
> Professor of Biochemistry and Chemistry
>
> Associate Chair of Biochemistry
>
> Department of Biochemistry
>
> University of Missouri
> 117 Schweitzer Hall
>
> 503 S College Avenue
> Columbia, MO 65211
> Phone: 573-884-1280
>
> Email: tanne...@missouri.edu 
> https://cafnrfaculty.missouri.edu/tannerlab/
>
> Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
>
> Office: Schlundt Annex 203A
>
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>

Re: [ccp4bb] Strategies for increasing Trp content of proteins

2022-02-19 Thread Wiener, Michael C (mcw2s) 
Dear Jack,

While we did this for Met (rather than Trp) insertion, (rather long ago) we 
used multiple sequence alignment to look for orthologs that had Met at 
positions where no Met was in our target protein. This worked to increase the # 
of methionines from two to eight, which enabled (rather) facile SAD phasing by 
Se-Met. Interestingly, the positions identified by MSA were often residues with 
little chemical similarity to Met (i.e., the six “winners” changed to Met were 
W, K, T, W, I, and Y).
(Chimento et al., NSB 10:394 [2003])

{Additionally, use of AlphaFold would likely also provide useful info on Trp 
placement.}

Regards,

-MW

--
Michael C. Wiener, Ph.D.
Professor of Molecular Physiology and Biological Physics
University of Virginia
PO Box 800886
Charlottesville, VA 22908-0886
434-243-2731 (office)
434-243-2730 (lab)

From: CCP4 bulletin board  on behalf of Artem Evdokimov 

Date: Saturday, February 19, 2022 at 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Strategies for increasing Trp content of proteins
Yes we chose the sites based on predicted or known structure, to be sure. No 
that was not a published thing, curse of commercial work :(

Happy to help on a private channel.

Artem

On Sat, Feb 19, 2022, 1:42 PM Tanner, John J. 
mailto:tanne...@missouri.edu>> wrote:
Casper: You make a good point about using 205 nm. I checked our system this 
morning and the wavelength is fixed at 280 nm (U9-L).

Artem: Did you consider secondary structure and/or amino acid residue type when 
choosing sites for mutagenesis? Is your work published?


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Casper Wilkens 
<5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>
Date: Saturday, February 19, 2022 at 2:19 AM
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins
WARNING: This message has originated from an External Source. This may be a 
phishing expedition that can result in unauthorized access to our IT System. 
Please use proper judgment and caution when opening attachments, clicking 
links, or responding to this email.

Is absorbance at 205 nm not a possibility, Jack?


Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering


Technical University of Denmark
[http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif]
Department of Biotechnology and Biomedicine
Søltofts Plads
Building 224
Room 028
2800  Kgs. Lyngby
c...@dtu.dk
www.bioengineering.dtu.dk/
https://www.cazypedia.org/index.php/User:Casper_Wilkens
https://twitter.com/protein_artist
https://www.researchgate.net/profile/Casper_Wilkens

Fra: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
på vegne af Artem Evdokimov 
mailto:artem.evdoki...@gmail.com>>
Sendt: 19. februar 2022 02:12:50
Til: CCP4BB@JISCMAIL.AC.UK
Emne: Re: [ccp4bb] Strategies for increasing Trp content of proteins

We have had success placing single trp on the outside of proteins, as weird as 
it sounds it works pretty well. If your protein is easy to express you can try 
a few choices.

If your protein is difficult to express, a fusion with a trp-positive