Protein expression

Theresa H via methods%40net.bio.net (by theresahsu8 from live.com)
Sat Jul 13 15:03:43 EST 2013


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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