palmitoylation

[Sean Patterson]

>seanpat at fmed2.uncu.edu.ar (Sean Patterson) wrote:
>:>In article <3A768700.BE5D1F43 at slac.stanford.edu>, Mark Bowen
>:><mbo at slac.stanford.edu> wrote:
>:>:Does anyone have experience with in vitro palimoylation?  I've tried the
>:>:reaction by adding palmitoyl CoA in DMSO to protein (25 kDa with 4 free
>:>:cys) in the presence of 1% octylglucoside.  This doesn't seems to be
>:>:working based on SDS-PAGE.
>:>:
>:
>:Lot's of experience. I mix 3H-palm-CoA (readily  soluble in water)
>:with my protein(s) of interest and incubate at 55C for an hour with
>:some detergent so there isn't a solubility problem after
>:palmitoylation.
>
>Sean, thanks for the info! Any chance you know what is temperature
>and pH dependence for this reaction? 55C is not great for many proteins
>(should be OK for this particular one, though). I am wondering if
>30C o/n at pH > 8 will do the trick.

I haven't (yet) done complete temperature or pH curves. The 
palmitoylation works at 37C, but is substantially faster at higher 
temps (surprise, surprise). I also suppose that a higher pH would be 
beneficial, and perhaps a lower one too.

>:What is your assay for successful palmitoylation of
>:your protein?
>
>This particular guy shows difference in mobility on SDS PAGE.

Out of curiosity - is it multiply palmitoylated or a small protein? I 
used to get a migration shift with palmitoylated GAP-43 that was 
temperature dependent - I only saw the shift when I ran the gels at 
high voltage, but not at high voltage in the cold room. I always 
thought that it was a hydrophobic interaction between the fatty acid 
and the polyacrylamide matrix.

>:Ditto that.  What is your protein and why do you need to 
>palmitoylate in vitro?
>:
>Sounds like the protein is SNAP25. It is 4X palmitated and strongly
>periferal membrane bound in vivo. It is easy expressable in E.coli
>in huge amounts, but without lipids it does not display all the properties
>of native protein.
>
>One thought on separation of lipidated protein from non-modified:
>dialyse out octyl glucoside -> lipidated form precipitates and
>since the protein is small and known to be largely unstructured
>the precipitate can likely be redissolved in detergent.
>
>Further fractionation to separate 1,2,3,4 palmitoyl might be
>possible with HIC (assuming there is a sorbent that will bind in
>the presence of detergent).
>
>         - Dima

Phase partitioning with Triton X-114 might also work, but I'm not 
sure how many palmitates would be necessary to push it into the 
detergent phase.

Sean


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