Re: [ccp4bb] SUMMARY: protease cleavage sites

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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From: "Winter, G (Graeme)" g {- dot -} winter {- at -} DL {- dot -} AC {- dot -} UK
Date: 2007-03-06

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Subject: Re: SUMMARY: protease cleavage sites
From: Rene Frank rf3 {- at -} SANGER {- dot -} AC {- dot -} UK
Date: 2007-03-06

Hi all,

Summary points for choice of proteases:

1. Most favoured protease is TEV protease for precision and cutting
efficiency (commercial source or expression vector widely available).
2. 3C/PreScission protease has not been used as frequently, but is
also strongly recommended.
3. Thrombin, factor Xa, enterokinase have had mixed reviews, with
some potential problems with secondary cleavage of your protein.
Using thrombin may require careful optimization including testing
multiple suppliers.
4. Other proteases: SUMO protease and TVMV protesase. The latter is
similar to TEV, but with a different specific excision site (vector
available from www.addgene.org).

5. Adding the same purification tag to your protease as you are using
to purify your protein is advocated for ease of removing the protease
afterwards.
6. For membrane and secreted proteins careful consideration of where
to put your N-terminal tags and protease sites after the signal
peptide is emphasized, as is avoiding putting a protease cleavage
site within predicted secondary structure.
7. Awareness of the excision site residues left after cleavage,
particularly for protease sites at the C-terminus.
8. Most of the proteases and their excision sites are nicely
tabulated here: http://stevens.scripps.edu/webpage/htsb/cleavage.html

I need to tag up to three subunits that I can selectively excise, so
I have decided to go for TEV, 3C/PreScission protease and TVMV.

Thank you for your helpful advice.

Rene

================================================
Dr R.A.W. Frank, PhD
Royal Commission for the Exhibition of 1851 Research Fellow

Prof Seth Grant Lab / Genes to Cognition
Wellcome Trust Sanger Institute
Hinxton
Cambridge CB10 1SA

Work Tel: 0044 (0)1223 834244 ext. 7318
Cell No.: 0044 (0)7870 208280
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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: XIA2 0.2.5 Released
From: "Winter, G (Graeme)" g {- dot -} winter {- at -} DL {- dot -} AC {- dot -} UK
Date: 2007-03-06

Next message:
Subject: OT: turbo frodo keys?
From: Sabuj Pattanayek sabuj {- dot -} pattanayek {- at -} VANDERBILT {- dot -} EDU
Date: 2007-03-06