We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) alpha-globin chain halo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile alpha-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant alpha-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) alpha-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 alpha-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified alpha-globin was also investigated. These results suggested that C. siamensis alpha-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. (C) 2014 Elsevier Inc. All rights reserved.