Q: fusion protein expressin

greenleaf at bchm.biochem.duke.edu greenleaf at bchm.biochem.duke.edu
Fri Dec 18 17:10:47 EST 1992


In article <1992Dec17.052937.12926 at nuscc.nus.sg> mcbzzs at nuscc.nus.sg (Zhao
Zhuoshen (Mr)) writes:
>Dear fellow-netters,
I normally collect 8 fractions 1ml each. But I can
>not get any proteins by this protocol. Are there any of you know what is
>wrong I did? Is there any people has a better protein expression and
>purification method? Any suggestion is appreciated.
>
>Thanks in advance.
>Jackson
>mcbzzs at nuscc.nus.sg
>
Your fusion protein is probably insoluble, so when you spin down the lysate,
what you want is in the pellet, in inclusion bodies.  You may have to resuspend
your inclusion bodies in an denaturant (see references by F.A.O. Marston for
ideas) to solubilize your fusion protein, then get rid of the denaturant.  I do
mine in 8M urea/50mM tris ph 8/ 50mM KCl/1mM edta/ 50mM DTT (to reduce
disulfides), homogenize in this, incubate for 1hr at 4degC, then dialyze into
avarious buffers without any salt (your protein may vary).  Then try the column
again.  My fusion still does not bind well to GSH agarose after this treatment,
but you may have better luck.  Hope this helps.


Andrea Skantar
Duke University Biochemistry Dept.
greenleaf at bchm.biochem.duke.edu 



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