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N-terminal degradation of Recombinant protein

Russell Wrobel rlwrobel at biochem.wisc.edu
Thu Feb 9 10:39:08 EST 2006

I'm part of a structural genomics group and we do high though put 
protein production in E. coli. N-term. tags can usually be cleaved off 
after protein purification with site specific proteases.  In our program 
we have had good luck expressing things with an N-term maltose binding 
protein (MBP) tag.  After purification we remove the tag using TEV 
protease.  There are several commercially available expression systems 
that rely on the cleavablity of solubility tags.

Good Luck, Russell

kirtan koticha wrote:
> I am trying to express a 9 Kd protein in pET vector
> with C-terminal Histag. I see little or no expression
> in half a dozen different strains ( with pLysS, with
> rare codons etc.) I have also tried a wide range of
> IPTG conc , induction times and induction
> temperatures. Western blot with His antibody shows
> intact His-tag but protein protein is getting
> degraded. Interestingly when expressed witha leader
> sequence (pelB) expression in the same strains is
> robust . Unfortunately It cannot pass through
> periplasm so pelB cannot be removed so the clone is
> useless. Have checked N-end rule for protein
> degradation (Varchasky 1992) and protein should be
> stable. Besides this the protein is mammalian, no
> peculiar features and water soluble.Attaching tag at
> N-terminal end is not preferable as it is the
> functional end of the protein. Any input is valuable,
> Thank you.
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