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Re: [ccp4bb] Tag, you're in! Flag, V5, etc
 

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CCP4bb <-- 2008 <-- February 2008 <-- 07 February 2008
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Subject: Re: Tag, you're in! Flag, V5, etc
From: James Whisstock James {- dot -} Whisstock {- at -} MED {- dot -} MONASH {- dot -} EDU {- dot -} AU
Date: 2008-02-07 		Next message:
Subject: Re: an over refined structure
From: Dean Madden drm0001 {- at -} DARTMOUTH {- dot -} EDU
Date: 2008-02-07


Subject: Re: Tag, you're in! Flag, V5, etc
From: Andrew Gulick gulick {- at -} HWI {- dot -} BUFFALO {- dot -} EDU
Date: 2008-02-07
If circumstances require a C-terminal tag, the intein system from New
England Biolabs has worked very well for us. The pTYB1 plasmid encodes a
fusion between your protein of interest, a viral intein (think "protein
intron") and a chitin binding domain. The fusion adsorbs to a chitin resin
and all other proteins are washed away. The intein is engineered to not
catalyze protein ligation but rather to release the protein in a
thiol-directed manner. To cleave your protein from the fusion, simply add
DTT, plug the column, and incubate overnight. The next day, "push" your
unbound protein off the column: native protein with no tag residues left!
The CBD-intein remain bound to the resin

You don't get a lot of protein (5-10mg is more common than 50mg) and it is
diluted to the volume of your column. On the other hand, it is pure enough
to go right into trials upon concentration.

At least that's been our experience.
Andy
(No affiliation with NEB)

On 2/7/08 4:33 PM, "James Whisstock"
wrote:

> Hiya
>
> We usually add all N-terminal tags with a TeV cleavable linker. C-terminal
> tags always seem a pain to remove cleanly, because most highly specific
> recognition sequences (such as TeV) take advantage of P rather than P'
> specificity so you are usually left with a five (or so) residue tail.
> Actually has anyone any neat tricks for C-terminal tag removal?
>
> J
>


--
Andrew M. Gulick, Ph.D.
-----------------------------------
(716) 898-8619
Hauptman-Woodward Institute
700 Ellicott St
Buffalo, NY 14203
-----------------------------------
Senior Research Scientist
Hauptman-Woodward Institute

Assistant Professor
Dept. of Structural Biology, SUNY at Buffalo

http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html
http://labs.hwi.buffalo.edu/gulick
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