Hydroxylamine for cleaving proteins?
kay at lyon151.inserm.fr
Thu Jan 18 05:14:04 EST 1996
marciam at unixg.ubc.ca (Marcia MacDonald) wrote:
>I am hoping to use hydroxylamine to cleave a phosphorylated protein but
>I have been unable to find any recent references on the procedure. Has
>anyoiaelse used this method? If so, will it interfere with the
>phosphorylated residues? Any information at all would be helpful.
I have used hydroxylamine a lot to cleave off the fusion part of
recombinant fusion proteins. The preferred cleavage site of
hydroxylamine is asparagine-glycine. According to "Methods in
Enzymology" (probably volume 47, quite old) it is thought that
the side chain of the asparagine lies over the glycine and at
basic pH there is cyclisation and cleavage by hydroxylamine.
Whether phosphorylation of ser or thr in an asn-gly-ser or thr
context will affect this, I do not know.You must stay under
pH 10, because this rearranges the asparagine.
My recombinant proteins were insoluble, so I had to do the
cleavage under denaturing conditions My recipe is:
Weigh out 28.4g of guanidine thiocyanate into a 50 ml plastic tube
and add water to a final volume of about 33 ml and leave to
dissolve overnight at 37°C. Not all dissolves, the solution is
supersaturated. Next day,put at 45°, add 3.55g of LiOH.H2O,
5.6g of NH2OH.HCl, 75 mg ofTris base and 0.4 ml of
2-mercaptoethanol (I find that this helps to dissolve the
insoluble protein). Adjust to a final volume of 40 ml.
Everything takes some time to dissolve, since the
solution is saturated. The final pH should be about 9.3.
If necessary, adjust wuth solid LiOH or NH2OH. Add to the
insoluble protein pellet and incubate at 45°C for 4 hr.Ammonia
is released, so from time to time I unscrew the cap to
release pressure.Dialyse, first at room temperature and then
at 4°C to remove the reagents.
It works well. I can usually get over 75% cleavage. For
one protein, I got some secondary cleavage, but the asn-gly
cleavage was still the major one.
Hope this helps
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