N-terminal degradation of Recombinant protein

kirtan koticha kirtank at yahoo.com
Wed Feb 8 09:27:34 EST 2006


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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