Successful recombinant production of Allochromatium vinosum cytochrome c' requires coexpression of cmm genes in heme-rich Escherichia coli JCB712.
Cytochrome c' from the purple photosynthetic bacterium Allochromatium vinosum (CCP) displays a unique, reversible dimer-to-monomer transition upon binding of NO, CO, and CN(-). This small, four helix bundle protein represents an attractive model for the study of other heme protein biosensors, provided a recombinant expression system is available. Here we report the development of an efficient expression system for CCP that makes use of a maltose binding protein fusion strategy to enhance periplasmic expression and allow easy purification by affinity chromatography. Coexpression of cytochrome c maturase genes and the use of a heme-rich Escherichia coli strain were found to be necessary to obtain reasonable yields of cytochrome c'. Characterization using circular dichroism, UV-vis spectroscopy, and size-exclusion chromatography confirms the native-like properties of the recombinant protein, including its ligand-induced monomerization.
Evers
Toon H
TH
Laboratory of Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
Merkx
Maarten
M
D002248:Carbon Monoxide / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002841:Chromatiaceae / Q000235:genetics* / Q000378:metabolism; D002844:Chromatium / Q000737:chemistry; D002850:Chromatography, Gel; D003486:Cyanides / Q000378:metabolism; D045305:Cytochromes c' / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry*; D008024:Ligands; D058619:Maltose-Binding Proteins; D008970:Molecular Weight; D009569:Nitric Oxide / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry
D002352:Carrier Proteins; D003486:Cyanides; D045305:Cytochromes c'; D008024:Ligands; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide