Hi,
You may find helpful suggestions in Kiyoshi Nagai's papers (mid 80ies) who did
this with haemoglobin (I could be wrong but I think he was the first to do
this).
Cheers,
Boaz
Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel
E-mail: [email protected]
Phone: 972-8-647-2220 Skype: boaz.shaanan
Fax: 972-8-647-2992 or 972-8-646-1710
________________________________________
From: CCP4 bulletin board [[email protected]] on behalf of Biswajit Pal
[[email protected]]
Sent: Monday, July 16, 2012 8:45 AM
To: [email protected]
Subject: [ccp4bb] 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
