Expression and purification of recombinant hemoglobin I from Lucina pectinata.
Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H2S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-PBAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of L-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.
Rosado-Ruiz
T
T
Chemistry Department, University of Puerto Rico, Mayagüez 00681, USA.
Antommattei-Pérez
F M
FM
Cadilla
C L
CL
López-Garriga
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins
D018076:DNA, Complementary; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D011994:Recombinant Proteins; D006418:Heme; C026236:hemoglobin I, non-mammalian