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
Biolabs,
in their latest catalog, admits to some low specificity of this
protease:
"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,

Arle
*************************************************
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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