Wade,
Our target biology group has mixed results with the TAT-fusion technology. I
have been able to show nuclear localization of TAT-rhodamine and polyArg
(9-mer)-rhodamine but only rarely do peptide fusions (N-terminal TAT and a
10-20 mer peptide) have any biological (functional) phenotype. The word on
the street is to denature proteins first and then apply them to the cells.
Good luck.
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Gordon Alton, Ph.D.
Lead Scientist
Assay Development and Analytical Protein Chemistry
Department of Informatics and Functional Genomics
Signal Research Division of Celgene
5555 Oberlin Drive
San Diego, CA 92121
Email: galton@signalpharm.com
Phone: 858-558-7500 x8252
Fax: 858-623-0870
WWW: http://www.signalpharm.com
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-----Original Message-----
From: Wade Edris [mailto:EDRISW@war.wyeth.com]
Sent: Tuesday, February 27, 2001 12:16 PM
To: Recipients of ABRF List
Subject: TAT-fusions
Dear ABRFers,
Our lab is currently purifying TAT-fusions for transduction studies. Our
test protein is TAT-Beta GAL. It is 6-HIS tagged, bacterially expressed,
and purifies to about 80% on a single pass thru Ni-NTA. 10 ng/ml turns
NO-pyranoside blue in a minute. The only trouble is, only 2 preps out of 5
ever showed any transducing ability!!! I don't know if the TAT leader is
degraded in any way. we will check this out by sequencing and MALDI. Does
anyone have any experience with these TAT-fusions? It seemed to me that the
preps that actually transduced cells had a precipitate in them (a
light-scattering haze). How does TAT work? does it monomerically thread
its way across membranes dragging its fusion partner along or is it some
kind of aggregation effect? I recall that aggregation of positively-charged
large molecules is necessary to transfect DNA (Ca-phos precipitation) and
the TAT leader is an 11 AA sequence with 8 basic residues. I guess I have
also heard that an 8-Arg pep!
tide leader can do the same thing as native TAT. any help will be greatly
appreciated.
wade
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