Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.

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.

Molecular cloning and expression of α-globin and ß-globin genes from crocodile (Crocodylus siamensis).

The first report of complete nucleotide sequences for α- and ß-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and ß-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the ß-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and ß-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.