Overexpression of a protein

cwilliams dwillims at mail.tcd.ie
Fri Oct 27 04:49:09 EST 1995


>---------- Forwarded message ----------
>Date: Tue, 17 Oct 1995 17:23:34 -0400 (EDT)
>From: Natalie Lu <tlu at welchlink.welch.jhu.edu>
>To: methods at net.bio.net
>Subject: Overexpression of a protien (fwd)


>I have problems to overexpress my protein. The gene (2kb) was 
>successfully cloned into pET21-a(+) vector. The 
(deleted)
I am also having a few problems expressing from a pET vector. I am trying
to express a membrane protein in E.coli. Using IPTG inducible, amp based
vectors induction proves lethal to the cell, some protein can be 
recovered (GST fusion, 6xHis) but is mostly proteolysed. So I moved on
to try pET-24d (kan based) however the cells harbouring the recombinant plasmid grew
even better than those with the parent plasmid and induction with IPTG
had no effect on growth (I checked the cells retained the correct plasmid)
so I plated (the cells are BL21 pLys S) on chloramp/kan plates +/-
IPTG. Novagen say pLys S cells retaining the ability to express should
not form colonies on + IPTG plates (Does anyone know why this is so?)
and it appears while those with the
nonrecombinant plasmid did not form colonies those with the recombinant
plasmid did. I retransformed but all transformants tested also grew +IPTG.
I think the cells are OK because a with different vector (also T7 based
but not pET) induction proves toxic.
Could it be I am getting leakly expression and hence only selecting 
clones without the ability to make protein? Why if so does there appear
to be such a high mutation rate ? Could there be a problem with the 
vector? Would it be worth trying BL21 pLys E?  
I would be most grateful for any advice.

Cara Baker
Biochemistry Dept. T.C.D.
e-mail : crbaker at mail.tcd.ie



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