aggregating protein not going into SDS-PAGE gel

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Oct 31 19:16:22 EST 2001


> I have a relatively hydrophobic protein: 73KDa, pI 9.5, which is proving
> very hard to purify, and harder to solubilise. It expresses well in
> codon-biased bacterium (as is relatively high GC) as a His tagged, MBP
> tagged or Nus tagged. The less soluble His-tagged fusion is the most
> difficult.

Did you try MBP-cleavage sequence-protein-His combo ? Worked extremely well
for us.

> When fresh bacterial extract is run an SDS-PAGE gel, the protein bands are
> clearly evident. However following freezing the protein will not enter the
> gel under normal conditions (I thought initially that it was degrading).
> However if the sample is not boiled it will enter the gel, and a build up
> of charge at the top of the gel appears to cause melting patterns, and I
> always see staining in the junction between the stacking and running gel.
> Alternatively if you raise the SDS in the boiling mix/sample to 10%, and
> boil the protein will enter the gel.  However, the ability of the protein
> to enter the gel; is also concentration dependant - if for example 500ml
> rather than 300ml is loaded no protein will enter under any conditions.

I am not sure what you mean when you say 500 ml. Did you mean to say
microliters ? Even so, 500 u is mighty high...
Did you try precast gradient or even single-concentration gels ? They might
be better in terms of protein not entering.

>So,
> aggregation is heat, cold, and concentration dependant.  I want to use the
> protein for immunisations.  The protein solubilises very well in Urea or
> guanidine but as soon as dialysed precipitates.  10% SDS at the

Well, if you're using it for immunizations, you don't really need to refold
it - correct ? Consider detergents. With a HIS tag you can run your
separation in urea, on an IMAC, then dialyze the urea out and the detergent
of choice in. If you can get away with it - great. Also try non-detergent
sulphobetaines (NDSBs).

Can you chop the protein up with proteases and figure out a good sequence to
immunize against ? Or you must have the whole thing ?

> denatuturation step assists to some extent.  Sometimes the precipitate is
> flaky sometimes clear and gelatinous (sticks to dialysis membranes).  I
> also cannot elute the protein from the gel once I have got it in. If
anyone
> has any ideas on how I can get this protein into the gel and
> concentrate/dialyse it such that it doesn't precipitate please let me
know.

Another idea would be to try glycerol or ethylene glycol in your running
buffers. Also consider octasulphonylsucrose (although it may do more harm
than good, often).

Hope this helps.

A.






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