ds4 at st-andrews.ac.uk
Wed Jul 3 12:03:01 EST 1996
My project is officially finished but I just can't stop fiddlin'.
Not content with my active peptide I now want to know it's precursor.
We try and do an affinity-purification of the prepropeptide from
crab haemocytes using an antibody against the active peptide. The
problem I expect to cause most difficulties is the presence of
proteases iun the cellular extracts which will spontaneously cleave the
prepropiece off the peptide so all we get is the active thing. I heard
of weird and wonderful formulations of enzyme inhibitor cocktails, but
as my remaining time is rather short, i prefer to try the quick and
dirty way first. Now I have seen in a paper by Hara & Yamakawa (Biochem. Biophys. Res. Com. 220, 664-669) that these authors have extracted recombinant
Moricin (an antibacterial peptide) expressed as a fusion protein in e. coli
by disrupting the cells with 4 M guanidine hydrochloride in 80% acteic
acid. This should abolish all protease activity. The authors cleaved
their peptide out chemically and found that it worked fine. I really
do not know what the effect of guanidine HCL on proteins is and
whether it might be used to purify a precursor protein or whether it
would simply destroy the original structure or shape of that molecule.
Can anybody enlighten me? Anyone ever tried such a thing? Sorry if I
sound far out, I freely admit my ignorance in these matters.
Gatty Marine Laboratory Fax: (0)1334-463443
University of St. Andrews e-mail: ds4 at st-and.ac.uk
St. Andrews Fife KY16 8LB
(Opinions expressed here are my own)
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