Expression and purification of recombinant hemoglobin I from Lucina pectinata

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to fo rm the ferric sulfide complex needed to transport H2S to the bacterial endo symbiont. To further study HbI, expression studies of this protein were per formed in Escherichia coli. This is the first time that the recombinant HE was produced using a recombinant DNA expression system. Hemoglobin I cDNA w as amplified and cloned into the TOPO-P-BAD expression vector, which contai ns a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequen ce analysis was carried out in order to ensure that the insert was in the c orrect reading frame for proper protein expression in E. coli. The expressi on of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-ar abinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombin ant protein was performed under native conditions by affinity chromatograph y using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinan t protein presented a shift from the Soret band at 413-435 nm, indicating t he presence of the heme group in the adequate amino acid environment of HbI These results indicate that recombinant HbI from Lucina pectinata can be s uccessfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.