help

Matthew Parker MatthewP at microbio.uab.edu
Sat Jan 23 16:29:58 EST 1999


yuzhang88 at HOTMAIL.COM ("yu zhang") wrote:

>Hi,everyone:
>     I cloned a IFN gene into pMAL-C2(NEB)Vector.The fermentation 
>indicated that the fusion protein was expressed,we purified IFN protein 
>by affinity chromatography with Amylose Resin. But it's not going on 
>smoothly.There were three problems with us:   Fist, the collection was 
>unclear after eluted the fusion protein from column with 10mM maltose.  
>Second,we want to get IFN by cleavage the collection by Factor Xa,and 
>then,use sephacryl s-100 to purify it. But,proteins including 
>uncleavaged fusion protein(60KD),MBP(40KD),IFN and other unknown 
>proteins are all in the first peak. Another peak with a little shoulder 
>was pure MBP showed by SDS-PAGE.  Third,only about 30% of the fusion 
>protein was cleavaged by Factor Xa.We have checked our sephacryl s-100 
>column with standard protin and the result showed that the column was 
>O.K. 
>      We would appreciate if you can give us some advice,thanks.
>
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Yuzhang,

	I think that your fusion protein may be aggregating, which
might be the source of the turbidity.

	I have had bad experiences using the New England Biolabs
maltose-binding-protein fusion kit, and I would not recommend it to
anyone. First of all, as you observed, it is difficult to fully cleave
the fusion protein unless you use a large amount of the factor Xa
protease (and it is expensive). Second, this protease has a tendency
to cleave at other sites, even those which are only similar to the
"specific" site it is supposed to recognize. Third, the amylose resin
(which is also expensive) can only be used a few times for purifying
the fusion from cell lysates, because amylases present in E. coli chew
it up.

	I don't know what to tell you. Maybe you could look into using
other fusion protein systems, for instance a histidine "tag." I also
remember reading about another system which employs some sort of tag
which is used to purify the fusion using affinity chromatography. The
nice part is that the protease which is used to cleave the fusion also
contains this tag, so the protease can be removed (and recovered)
after cleavage. I can't remember which company makes it, but if you
are interested I will look it up and let you know.

	Good luck!

	Matt




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