Expression and purification of recombinant hemoglobin I from Lucina pectinata.

摘要: Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric complex needed transport H2S bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is first time that recombinant HbI was produced using a DNA system. cDNA amplified and cloned into TOPO-PBAD vector, which contains fusion tag six histidine residues (6XHis tag). Plasmid clone sequence analysis carried out order ensure insert correct reading frame for proper E. The optimal when induced 5 hr 0.002% L-arabinose as detected by Western blot analysis. proto-porphyrin group inserted HbI. Purification heme-bound under native conditions affinity chromatography Ni-NTA Probond resins. sodium dithionite-reduced presented shift Soret band at 413-435 nm, indicating presence heme adequate amino acid environment These results indicate can be successfully expressed prokaryotic system retaining its activity toward reduction, oxidation, ligand binding.