[Q] Best Protease for Tag Removal

Michael Oda mnoda at lbl.gov
Tue Nov 21 15:25:11 EST 2000


Hi,

> :We have also been considering placing the amino acids Asp-Pro with their 
> :acid labile peptide bond between the His tag and the target protein. 
> :Does anybody have any experience with this particular peptide bond and 
> :its acid lability? Fortunately our protein is rather durable and folds 
> :back to a native conformation after 3M Guanidine treatment and appears 
> :to be somewhat acid stable.
> 
> No idea how efficient it is. Perhaps worth trying but I'd be sceptical of
> this approach. Acid+heat will, albeit with lower efficiency, hydrolyse 
> other peptide bonds; you might end up with a mess that will require 
> further purification; also, you might already have Asp-Pro in your 
> sequence. 

We have done a bit of homework regarding our protein and found it 
doesn't have an asp-pro bond but does have glu-pro bonds (2). Thinking 
that this bond may also be acid labile we did a survey of conditions and 
found that our protein was reasonably stable (~80% full length) in up to 
50% formic acid at 37° C for four days. Now the question is, is that 
sufficient for bond hydrolysis?

We have searched for the use of this bond in applications beyond peptide 
sequencing and haven't encountered any. I was hoping that someone out 
there had used this before and had created a more gentle condition than 
50% formic acid, four days @ 37°C, saving me the great effort of 
empirically elicidating the minimum acidic environment necessary for 
bond hydrolysis.

Thank you,
Michael Oda

-- 
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Michael Oda, Ph.D.                     |  Phone: (510) 486-4088      
Donner Labs                            |    Fax: (510) 486-4750       
Lawrence Berkeley National Laboratory  |
Berkeley, CA  94720                    |






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