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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