Expression, purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides.
The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.
Potter
Christopher A
CA
Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Ward
Alison
A
Laguri
Cedric
C
Williamson
Michael P
MP
Henderson
Peter J F
PJ
Phillips-Jones
Mary K
MK
D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011494:Protein Kinases; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013997:Time Factors
D001426:Bacterial Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; C072722:RegA protein, Bacteria; C091081:RegB protein, Rhodobacter; D011494:Protein Kinases