Thorsten Schmidt wrote:
>> Dear Sir or Madam!
>> I have big Problems with Novagen`s HisTag Protein Purification-Kit.
>> I followed strikly Novagen´s purification protocol but the problems begin
> with the
> total protein. Instead of the predicted 50-100 mg, I get only 2 (!).
> Changes in the protocol (addition of Lysozyme, 0,1 % Triton) increases the
> total protein amount only to 30 mg.
> This leads to my first question:
> What can I also do to increase the total protein yield?
Expression levels of recombinant proteins can vary widely even when
using the same expression vector and system. For example, proteins
whose N-terminal sequences contain several grouped proline residues
often express poorly. Even for identical polypeptide sequences,
expression can vary depending on which of the redundant nucleotide
codons are used (many mammalian proteins do not express optimally in E.
coli because E. coli has different codon preferences).
> May Lysozyme or Triton lead to problems with the HisTag purification?
Lysozyme shouldn't, and I doubt Triton will either, but I have no
personal experience with either.
> Although SDS-PAGE analysis showed that the target protein is expressed, I
> got no target protein (0,04 mg instead of 20 mg!).
> Why is it impossible to purify my target protein?
While I have not done this myself, I know the experience of my Amgen
colleagues is that purification of His-tagged proteins on the Ni resin
fails fairly often, either because the protein hardly binds at all, or
because many other proteins are eluted along with the one of interest.
The view here is that His-tag purification of FOLDED proteins seldom
works, probably because the tag is sterically restrained from binding
well to the resin. You may have better success if you explicitly run
your Ni column under conditions where the protein will be denatured
(perhaps adding urea or guanidine-HCl), but then you will have to refold
the protein in a separate step. If you are hoping to purify a folded
protein using the Ni resin, you might want to try adding a spacer
sequence between your protein and the His tag so it is more exposed.
The view here is also that protein binding to the Ni resin is less
specific than one might wish. It seems that often other proteins are
bound and elute with the tagged protein. Presumably these other
proteins accidentally contain clusters of histidines.
'Hope this helps. Good luck.
John Philo, Protein Chemistry
Amgen Inc., Thousand Oaks, CA
jphilo at amgen.com
*** Disclaimer: These are the opinions of the poster not Amgen Inc.***