patrick at howard.genetics.utah.edu
Mon May 13 07:49:56 EST 1996
On Mon, 13 May 1996, Jurgen Vanhauwe wrote:
> Lipofection seems to me the only way to deliver my cargo because the
> proteins that I want inside in an active form need to be
> posttranslationally modified. Then also the question raises if the two
> proteins will interact after lipofection. Is my host (E.coli) going to
> degrade these intruders or shouldn't I worry about that? Any advice?
> Experience in lipofection?
My next question is do you need to use E. coli? They require a bit more
work to maintain but it is still rather simple to use a cell line...and
cell lines are very amenable to lipofection. As for posttranslational
modification, am I to assume that they are modified prior to insertion?
Or are you expecting E. coli to do it? If the latter, then there might
be an additional problem in that E. coli may not modify them or may
modify them in a way entirely different than you expect.
An alternative to cell lines (HeLa, NIH 3TC, etc) is yeast (S.
cerevisae). You could temporarily remove their chitinous cell wall,
leaving a rather fragile spheroplast, with only a cell membrane,
lipofect, and let them recover their cell wall. Yeast is essentially as
simple to deal with overall as E. coli...just slightly different agars
and techniques. Yeast is also far more likely to properly modify a
protein, or accept a modified protein, in a way you might wish than bacteria.
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