Abstract
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.
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