Re: [ccp4bb] Heme incorporation in expressed protein
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: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Biswajit Pal [p...@ccmb.res.in] Sent: Monday, July 16, 2012 8:45 AM To: CCP4BB@JISCMAIL.AC.UK 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
Re: [ccp4bb] Heme incorporation in expressed protein
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
Re: [ccp4bb] Heme incorporation in expressed protein
Incomplete heme-incorporation in heme proteins expressed in E. coli. is a common issue. Check out the following paper for an easy method, which, in our experience, has solved all our issues regarding this problem. This has worked really well for many different heme-binding proteins (Cys / His ligated, mammalian / bacterial proteins) Protein Expr Purif. 2010 Sep;73(1):78-82. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli. http://www.ncbi.nlm.nih.gov/pubmed/20303407 Jawahar Sudhamsu, Ph.D. 1 DNA Way, MS-27 Dept. of Structural Biology, Genentech Inc. South San Francisco, CA - 94080 On Mon, Jul 16, 2012 at 4:19 PM, Ho Leung Ng h...@hawaii.edu wrote: 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
[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
