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)
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
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 Stre
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 o
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
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
Sølto
