Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli

• Published in: Protein expression and purification Vol. 73; no. 1; pp. 78 - 82
• Main Authors: Sudhamsu, Jawahar, Kabir, Mariam, Airola, Michael V., Patel, Bhumit A., Yeh, Syun-Ru, Rousseau, Denis L., Crane, Brian R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-09-2010
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Escherichia coli - metabolism; Ferrochelatase; Ferrochelatase - biosynthesis; Ferrochelatase - chemistry; Ferrochelatase - genetics; Ferrochelatase - metabolism; Free-base porphyrin; Heme - metabolism; Heme incorporation; Heme proteins; Hemeproteins - chemistry; Hemeproteins - genetics; Hemeproteins - metabolism; Histidine - genetics; Histidine - metabolism; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Spectrum Analysis; Spectrum Analysis, Raman; Heme incorporation; Ferrochelatase; Heme proteins; Free-base porphyrin
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.03.010

Over-expression of heme binding proteins in
Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in
E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.