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Cleavable C-terminal fusion proteins

Arle Kruckeberg arle at biovax.leeds.ac.uk
Thu Nov 23 18:23:45 EST 1995

In article <J.L.Hobman.74.000C9C9C at bham.ac.uk>
J.L.Hobman at bham.ac.uk (Jon Hobman) writes:

> I wonder if anyone knows of an expression vector that has a C-terminal tag 
> (such as 6x His) that has a specific protease cleavage site between your 
> protein and the tail. I've found loads of N-terminal cleavable fusion tags but 
> I want to tag a periplasmic metal binding protein which has a cleaved leader 
> sequence (hence no N-term tags) and we are concerned that a big nickel binding 
> hexapeptide might well alter dramatically the metal binding propeties of the 
> protein (hence removal of the tag).

I tagged my protein with a hexahis carboxyl terminus, preceded by a
Factor Xa
cleavage site, but the protease also cleaves my protein. New England
in their latest catalog, admits to some low specificity of this
"Factor Xa cleaves after the arginine residue in the amino acid
sequence Ile-(Glu or Asp)-Gly-Arg. However, proteins can sometimes be
cleaved at
other arginines, depending on the context...A number of the secondary
sites have been sequenced that show cleavage following gly-arg [my
protein's Achilles heel]. There is also indirect evidence that suggests
Factor Xa can cleave at lysine residues...There seems to be a
correlation between proteins that are unstable in E. coli and those
that are cleaved by Factor Xa at secondary sites..." 
Enterokinase is popular around here, and a recombinant tobacco etch
virus protease with a cleavage site EXXYXQS/G has been described. It is
hexahis-tagged, and is described for use to cleave away GST tags from
your protein and then get rid of the protease on a nickel column (Parks
et al, Analyt. Biochem. 216:413 (1994)). You could swap the hexahis bit
for a GST tag on the TEV protease, and ....

Good luck,

a.k.a. Dr. Arthur L. Kruckeberg
 Department of Biochemistry and Molecular Biology
University of Leeds        phone  +44 +113 2333172
Leeds LS2 9JT              FAX    +44 +113 2333167
Great Britain              arle at biovax.leeds.ac.uk

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