Autoinduction, purification, and characterization of soluble a-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli Thai Kabbua a,d , Preeyanan Anwised a,d , Atcha Boonmee b,d , Bishnu P. Subedi c , Brad S. Pierce c , Sompong Thammasirirak a,d,⇑ a Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand b Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019, USA d Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand article info Article history: Received 19 May 2014 and in revised form 19 August 2014 Available online 28 August 2014 Keywords: Heme Disulfide bond Electron paramagnetic resonance Circular dichroism Cyanide binding Nitric oxide binding abstract We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) a-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system with- out addition of exogenous heme. This is the first time that heme-bound crocodile a-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant a-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) a-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 a-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified a-globin was also investigated. These results suggested that C. siamensis a-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 assem- bly of crocodile hemoglobin. Ó 2014 Elsevier Inc. All rights reserved. Introduction Hemoglobin (Hb) 1 is the quantitatively predominant heme con- taining protein or hemoproteins. Its primary function is to bind, transport, and release molecular oxygen (oxygen carrier) [1,2]. Native Hb comprises four polypeptide subunits known as globin domains, two identical a- and two identical b-globin chains. Each chain contains a prosthetic heme b (protoporphyrin IX) group. The iron bound within each heme cofactor can bind small molecules in either the oxidized (ferric) or reduced (ferrous) oxidation states [3]. The heme iron is coordinated by a histidine residue where the 6th coordination site is open for the binding of various ligands [4]. Under physiologic conditions, the biosynthesis of a- and b-globin is normally equivalent. Endogenous or exogenous heme rapidly binds to the folded globin domains, either during translation or shortly thereafter [5]. A molecular system for Hb assembly of globin chains and heme groups was described by Graves et al. [6]. The newly formed a- and b-globin chains have to fold prior to associa- tion to form a relatively stable ab dimer. These dimers or the folded monomers then bind heme to form hologlobin (heme-bound) sub- units, which in turn rapidly self-associate to form the final holo-tet- ramer that is stable. By contrast, Hb globin domains lacking the heme cofactor (designated apoglobin) are very unstable, exhibiting rapid denaturation at ambient or elevated temperatures. As the sta- bility and function of Hb is very sensitive to proper (a 2 b 2 ) quaternary structure and full cofactor incorporation, it is important to elucidate the relevant intermolecular factors between individual a- and b-glo- bin chains which contribute to protein stability and proper function [5]. To date, structure–function studies of Hb have been based on http://dx.doi.org/10.1016/j.pep.2014.08.013 1046-5928/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Department of Biochemistry, Faculty of Science and Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen 40002, Thailand. Tel./fax: +66 43342911. E-mail address: somkly@kku.ac.th (S. Thammasirirak). 1 Abbreviations used: Hb, hemoglobin; IPTG, isopropyl-b-D-thiogalactopyranoside; CroHb, crocodilian hemoglobin; BPG, D-2,3-bisphosphoglycerate; TF, trigger factor; IMAC, immobilized metal affinity chromatography; CVs, column volumes; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; EPR, electron paramag- netic resonance; CD, circular dichroism; DTT, dithiothreitol; HbNO, nitrosyl-bound CroHb; H-NOX, Heme-Nitric oxide and/or OXygen binding; sGC, soluble guanylate cyclase. Protein Expression and Purification 103 (2014) 56–63 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep