[RASMB] HIS tags

[John Correia]

In 1999 Richard Thomas asked about His tags and the occurrence of
aggregation induced by their addition to a protein - search the rasmb
archive with a boolean His and tags to see that discussion..

This has come up again in a student's committee meeting discussion of
false positives in a two -hybrid system, and subsequent testing of in
vivo and in vitro pull down assays - (Yes Yes I'm telling them to do
AUC) - so I am revisiting the question with specific interest in finding
out about papers that report His tag artifacts, specifically aggregation
or folding effects of His tags....

Thanks in advance for any contributions.

I will share one set of observations with you to demonstrate this is a
universal concern - we have developed maltose binding protein constructs
with epitopes from the COOH tails of the beta-tubulin gene products for
the purpose of constructing standard curves when doing quantitative
western blot analysis on cell culture extracts.  Pure beta isotypes
don't generally exist for this purpose.  The tubulin COOH tails knocked
out maltose binding?????  The guess is the tails fold back like a
scorpion and disrupt folding or the binding site directly???

So next we added His tags & used those to isolate the fusion proteins -
have not yet tested them for aggregation effects - they do react with
the Ab's we have & the maltose binding protein domain has the simple
functions of providing weight so it runs reasonably on an SDS gel and a
measurable absorbance for concentration determination.

Maybe the question is about modular proteins that lack cooperativity!!

-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
 fax (601) 984-1501                             
 email address: jcorreia at biochem.umsmed.edu     
 homepage location: http://biochemistry.umc.edu/correia.html  
 dept homepage location:    http://biochemistry.umc.edu
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