about pET vector!

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Jun 13 02:22:19 EST 2000


> Boss wrote:
> 
> Hello,
> 
> I've tried some expression experiments using a pET28 vector, to
> express a certain enzyme. And in my operation, I've modified the
> vector by adding a leader sequence to it, because I want to locate my
> protein in periplasm. Now I found the location failed. Furthermore,
> the expression level had been quite low. My protein only occupied
> about 8% of the total proteins.
> Here's my trouble. would someone please give me some advice?
> Thanks a lot.
> 
> Chen


Hi Chen,
Maybe the localization didn´t work because the protein was not soluble.
In many cases, recombinant proteins form inclusion bodies (insoluble
masses of kinetically trapped folding intermediates). If that is the
case for your protein, it is likely that no transport occurs.

In addition, your expression level (8% of total protein) is NOT LOW. NOT
AT ALL! Be happy with it, many proteins do not express in levels higher
than 1%.

Frank






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