Clarification of Purification GST fusion proteins

David J Benz biotech at ni.net
Tue Jan 16 14:15:12 EST 1996


I realized that I made a confusing contribution to a thread last week, and
I wanted to clarify. If your protein is a GST fusion, then obviously it
needs to pass over the glutathione column in relatively native
conformation, so glutathione can be recognized at the active site of GST
and bind. However, if one uses the hisTag system from the pET vector line,
then the fusion protein can be somewhat denatured as it passes ovet the
column, because the source of the affinity is just the electrostatic
attraction between the N-terminal stretch of His residues in the protein
and the Ni-NTA resin. Tertiary structure does not have to be exact in this
case.  Thus, one advantage of the HisTag is that proteins in 8M
Guanidine-HCL can be passed over the column and afinity purified. I think
that a GST fusion protein in 8M Gu-HCL would not stick, since the binding
site tertiary structure is compromised.

David



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