N-term tags in E.coli

Eric C. Anderson anderson at pharmdec.wustl.edu
Mon Jun 12 17:55:40 EST 1995


In article <3rdaqj$ib at lyra.csx.cam.ac.uk>, cdw1000 at mole.bio.cam.ac.uk
(Christopher Walentas (Bioc)) wrote:
> Anyone have any problems/ experience with mucking about with
> the N-term of proteins to be expressed in Ecoli?  I've got a nice
> large globular protein (100 kDa homodimer) which expresses very
> well from a Ptac.  When I went to put a tag on the front, the whole
> thing died-- no expression that I could detect (activity, we ran
> out of Abs).  The tag itself is just after the Met, and so it has
> been suggested that it might be a processing thing (although aren't
> there some over-expressed proteins which don't get fully processed
> wrt their Met getting removed).  Does it really matter?  I'll
> try an invitro coupled translation on Monday, but I think it might
> be the bugs that don't like the new and improved version.

just thought that i'd add the experience of our lab in mucking about with
various ends of proteins.  one of the proteins that our lab works with has
a highly conserved C-terminal region.  if more than the last 4 amino acids
are deleted from the protein, or if any sort of tag is added to it (the
6xHis-Tag is the one which has been checked), the protein loses activity
completely.  putting a tag on the N-terminus works just fine (as the
activity assays running on the HPLC right now will hopefully continue to
prove).

another protein which has been studied in this lab had the opposite
problem.  when the His-tag was added to the N-terminus of this protein it
died.  tacking on the other end however resulted in almost full recovery
of protein activity when compared to wild type.

also...don't forget to make sure that the tag, protein and promoter are in
the same reading frame.  i know this is obvious but it almost got me once.

good luck,

eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)362-7058 (FAX)
660 s. euclid box 8103                           
st. louis, mo 63110

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