Protein expression

[Imran khan]

If the cells are dying that means the protein is toxic to the cells and could result in cell stress related proteolysis. Using a different strain of E.coli could help. does the his- antibody give any band at all? 


________________________________
 From: Hiranya Roychowdhury <hroychow from nmsu.edu>
To: Theresa H <theresahsu8 from live.com>; "methods from net.bio.net" <methods from magpie.bio.indiana.edu> 
Sent: Sunday, July 14, 2013 5:27 AM
Subject: RE: Protein expression
 

It will be important to know what the base changes did to the protein, especially since it is otherwise homologous.  There is a possibility that the base changes caused leakiness when integrated into the bacterial membrane.

Hiranya S. Roychowdhury, Ph.D.
Associate Professor
Health & Public Services
NMSU-Dona Ana Community  College
575 527 7725 (office)

________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] on behalf of Theresa H [theresahsu8 from live.com]
Sent: Saturday, July 13, 2013 2:03 PM
To: methods from net.bio.net
Subject: Protein expression

Dear all

I am trying to express a 90 kDa bacterial membrane protein in E. coli. The gene is nearly identical to the originating organism except with a few bases due to restriction site choice at the 5' end and His-tag at the 3' end. The expression is by T7 polymerase in a pET vector.  I have tried several strains and temperatures but I do not see my protein using Western blot, the antibody targeted against His-tag. When I induce with 1 mM and 0.5 mM IPTG, the cells stop grow based on OD reading over 12 hours.

Any help to solve the problem will be appreciated.

Thank you.
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