I was able to express a heme protein by inducing and expressing at room temperature and using a promoter weaker than T7 (can't remember the exact one right now). The key was to slow down the rate of protein production to allow heme incorporation. You might try using less IPTG too.
Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu On Mon, Jul 16, 2012 at 1:00 PM, CCP4BB automatic digest system <lists...@jiscmail.ac.uk> wrote: > > Date: Mon, 16 Jul 2012 11:15:59 +0530 > From: Biswajit Pal <p...@ccmb.res.in> > Subject: Heme incorporation in expressed protein > > Dear all, > Sorry for non-crystallography related question. > We are trying to express an eukaryotic heme protein in E. coli. We are able > to express it in soluble form. When we use 5-Aminolevulinic Acid, E. coli > becomes brown. However, after cell lysis the soluble protein contains no heme > and the pellet is brown. If we extract the pellet with detergent (Triton > X-100 and Tween-20) the color comes in the supernatant, but there is no > protein of our interest. These eventually indicate that the protein and heme > are getting expressed/synthesized, but heme is not getting incorporated in > the expressed protein. We would like to get this protein in heme-bound > holo-form. > Any suggestion to trouble-shoot this problem would be highly appreciated. > Replies can be sent to me directly and I will post a summary on a later date. > Thanks in advance, > Sincerely yours, > Biswajit Pal > CCMB, Hyderabad, India
