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 <[email protected]> on behalf of Artem Evdokimov
<[email protected]>
Date: Saturday, February 19, 2022 at 2:28 PM
To: [email protected] <[email protected]>
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.
<[email protected]<mailto:[email protected]>> 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 <[email protected]<mailto:[email protected]>>
on behalf of Casper Wilkens
<[email protected]<mailto:[email protected]>>
Date: Saturday, February 19, 2022 at 2:19 AM
To: [email protected]<mailto:[email protected]>
<[email protected]<mailto:[email protected]>>
Subject: [ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of proteins
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Is absorbance at 205 nm not a possibility, Jack?
Casper Wilkens
Asst. Prof.
Structural Enzymology & Biorefining
DTU Bioengineering
Technical University of Denmark
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Fra: CCP4 bulletin board <[email protected]<mailto:[email protected]>>
på vegne af Artem Evdokimov
<[email protected]<mailto:[email protected]>>
Sendt: 19. februar 2022 02:12:50
Til: [email protected]<mailto:[email protected]>
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.
<[email protected]<mailto:[email protected]>> 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: [email protected]<mailto:[email protected]>
https://cafnrfaculty.missouri.edu/tannerlab/
Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
Office: Schlundt Annex 203A
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