help

yu zhang yuzhang88 at HOTMAIL.COM
Sat Jan 23 09:30:11 EST 1999


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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