His-tagged proteins; Ni-NTA; contamination

davesmith at bioch.tamu.edu davesmith at bioch.tamu.edu
Wed Jan 3 19:25:40 EST 1996


We have gained some experience recently with Histidine-tagged
proteins.  We have several versions of tagged proteins in our
lab--N-His, C-His, and even internal tags.  In reading a recent post,
I noticed a reference to a company in Germany, "Connex", which sells a
monoclonal Ab to oligo-His.  Another post mentioned an antibody
provided by Qiagen.  We have no experience with the former, although
we will pursue it....thank you Russell Linscott!
With regard to Qiagen, I can vouch for the Ni-NTA-alkaline phosphatase
conjugate.  It recognizes all three of the versions of our protein
mentioned above, although with less overall intensity than we
expected.  The conjugate also cross-reacts with hen egg white
LYSOZYME, but NOT carbonic anhydrase (in the pre-stained low mw
markers we use for blotting) as suggested in the protocol and
literature provided by Qiagen.  If you use this conjugate on any form
of soluble bacterial extracts or supernatants you will also find that
it recognizes a ~20 plus kDa protein VERY well.  Not surprisingly the
E. coli  protein which often contaminates Ni-NTA purified proteins is
the same which cross reacts with the conjugate.  It's called SlyD from
sensitivity to lysis (by phiX174).  The protein is a member of the
peptidyl prolyl isomerase family and has a histidine-rich C-terminal
tail.  This explains its innate ability to be bound by Ni-NTA.  Qiagen
is aware of the source of the problem.  SlyD happens to be one of the
projects in our lab.  SlyD mutants of E.coli have been made and
"offered" to various companies---time will tell if they become
commercially available.  We have not used Qiagen's very expensive
His-tag antibody--as it is specific to the fortuitous amino acids
(only 6 of which are histidines) fused at the N-terminus of proteins
expressed from the pQE vector series.
Sorry this post is so long, but I thought there might be lots of folks
interested in the topic.  That's my 2 bits worth.

Dave Smith
(davesmith at bioch.tamu.edu)
lowly grad student




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