title: Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.
abstract: We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin.
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Thai
authors.initials: T
authors.lastname: Kabbua
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Preeyanan
authors.initials: P
authors.lastname: Anwised
authors.affiliation: Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Atcha
authors.initials: A
authors.lastname: Boonmee
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019, USA.
authors.forename: Bishnu P
authors.initials: BP
authors.lastname: Subedi
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019, USA.
authors.forename: Brad S
authors.initials: BS
authors.lastname: Pierce
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address: somkly@kku.ac.th.
authors.forename: Sompong
authors.initials: S
authors.lastname: Thammasirirak
mesh_terms: D000489:Alligators and Crocodiles; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004926:Escherichia coli; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013057:Spectrum Analysis; D055542:alpha-Globins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D006454:Hemoglobins; D055542:alpha-Globins; D006418:Heme
keywords: Circular dichroism; Cyanide binding; Disulfide bond; Electron paramagnetic resonance; Heme; Nitric oxide binding