Recombinant PAS-heme domains of oxygen sensing proteins: high level production and physical characterization.
Details of a high-level recombinant production method for the heme-PAS domains of heme oxygen sensing proteins from Sinorhizobium meliloti (Sm) (formerly Rhizobium meliloti, Rm), Bradyrhizobium japonicum (Bj), and Escherichia coli (Ec) are described. Using a newly proposed, concise, and unambiguous naming system (also described here) these proteins are: SmFixLH(128-264), BjFixLH(140-270), and EcDosH(1-147). In addition, high-level production of BjFixL(140-505), the soluble full-length protein containing both heme (oxygen sensing) and kinase (catalytic) domains is described. Using an IPTG-inducible pET/BL21 expression system and a rapid, two-column purification has resulted in increased yields of 3- to 17-fold over literature values. The recombinant proteins are highly pure as judged by SDS-PAGE, MALDI-TOF mass spectrometry, and a UV-visible purity index. To our knowledge, this work includes the first mass spectrometry analysis of any PAS-heme protein and provides high-resolution confirmation of each protein's identity. These production and characterization improvements make possible future spectroscopic and dynamics studies designed to elucidate the intramolecular/interdomain signal that follows heme-domain oxygen dissociation.
Suquet
Christine
C
Department of Chemistry, Washington State University, Pullman, 99164-4630, USA.
Savenkova
Marina
M
Satterlee
James D
JD
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000201:enzymology / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006420:Hemeproteins / Q000096:biosynthesis / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016962:Sinorhizobium meliloti / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006420:Hemeproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010727:Phosphoric Diester Hydrolases; C464427:dosP protein, E coli