sferguso at kimbark.uchicago.edu
Fri May 6 06:22:27 EST 1994
In article <2ppr1iINNf3t at medicine.wustl.edu> HAVILAND at KIDS.WUSTL.EDU (David L. Haviland, Ph.D.) writes:
>I have a question for those using any of the fusion protein expression
>systems such as GST and the like. We are interested in setting this
>technology up in our lab. Our goal is not only to make the fusion peptides
>as a source of purified peptide by cleaving off the GST but also for use in
>immunization, and thatt's where my question lies.
>Ig GST is kept on the fusion peptide by eluting it off the column, can the
>GST serve as the carrier in immunizations, circumventing the necessity of
>coupling the peptide to a more traditional carrier like KLH
Yes, if by peptide, you don't mean some little oligopeptide. If it's really
short, the number of epitopes is limited and it would be better to have a
higher peptide load by having a high substitution ratio of peptide:carrier.
If you mean a stretch of aa's that's around 50 aa or longer, then a fusion
protein strategy should be fine.
from Pharmacia, but I have
>read in the literature (and heard from the director of the local hybridoma directed against fusion protiens carrying
>protein-A often have the immune response biased toward the Protein-A and
>not the peptide. AIs this phenomena been observed using GST or MBP based
Sorry your text above is all screwed up. Some little turd i don't know
tried to talk to me over the net while I was writing. His connect
message rearranged and deleted some text. Gotta remember to disable my talk
function when I'm on newsgroups!
Obviously, this will depend entirely on the peptide you want antibodies to.
The more foreign epitopes on your peptide of interest, the greater the immune
resonse to that part of the fusion protein. You _will_ get a good response
to the GST, regardless of what it's coupled to and the level of the response
to that portion of the fusion protein is largely unaffected by what is
coupled to it. The ratio of GST-specific:your peptide-specific antibodies
is a function of the immunogenicity of your peptide. This would be true with
standard carriers as well, however.
While the affinities/avidities of the antibodies to your protein will
obviously be different from those to GST, you can try testing the mouse
sera before you even bother going through the time and expense of analyzing
all the hybridomas that may be generated for nothing if the response to your
peptide is poor. Can you do a Western with good controls to see if you got
a decent response? If you can find a way to see if the response is good
_before_ you've gone through lots of time and money necessary for generating
monclonals and testing them individually. If your core is good (and a lot
of hybridoma cores aren't), they should be able to generate scores if not
hundreds of monoclonals from a couple of mice.
And assuming that they are good and can do preliminary screening for you,
they should be able to eliminate all the GST-reactive clones by ELISA, using
GST alone. You should provide them with purified GST protein (just transform
with vector alone and you'll get TONS of the stuff off the column) for this
>Thanks in advance,
>(also crossposted (I hope) on the bionet.immunology sub)
>+ David L. Haviland, Ph.D. Internet:"haviland at kids.wustl.edu" +
>+ Washington Univ. School of Med. A.K.A : The Compiler +
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