Abstract
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.
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REFERENCES
Alam, S. L., Dutton, D. P., and Satterlee, J. D. (1994). Biochemistry 33, 10337–10344.
Antommattei-Perez, F. M., Rosado, T., Cadilla, C., and Lopez-Garriga, J. (1999). J.Protein Chem. 18, 831–836.
Bashford, D., Chotia, C., and Lesk, A. M. (1987). J.Mol.Biol. 196, 199–216.
Cerda, J., Echevarria, Y., Morales, E., and López-Garriga, J. (1999). Biospectroscopy 5, 200–213.
Cerda-Colón, J., Silfa, E., and López-Garriga, J. (1998). J.Am.Chem.Soc. 120, 9312–9317.
Chomczynski, P. and Sacchi, N. (1987). Anal.Biochem. 162, 156–157.
De JesÚs-Bonilla, W., Cortés-Figueroa, J. E., Souto-Bachiller, F., Rodríguez, L., and López-Garriga, J. (2001). Arch.Biochem.Biophys. 390, 304–308.
Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995).J.Bacteriol. 177,4121–4130.
Hoffman, A. and Roeder, R. G. (1991). Nucl.Acid Res. 19, 6337.
Kraus, D. W. and Wittenberg, J. B. (1990). J.Biol.Chem. 265, 16043–16053.
Kraus, D. W., Wittenberg, J. B., Jing-Fen, L., and Peisach, J. (1990). J.Biol.Chem. 265, 16054–16059.
Newman, J. R. and Fugua, C. (1999). Gene 227, 197–203.
Read, K. R. H. (1962). Biol.Bull. 123, 605–617.
Read, K. R. H. (1965). Comp.Biochem.Physiol. 15, 137–158.
Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994). J.Mol.Biol. 244, 86–99.
Rizzi, M., Wittenberg, J. B., Coda, A., Ascenzi, P., and Bolognesi, M. (1996). J.Mol.Biol. 258, 1–5.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 1–3.
Silfa, E., Almeida, M., Cerda, J., Wu, S., and López-Garriga, J. (1998). Biospectroscopy 4, 311–326.
Tokishita, S., Shiga, Y., Kimura, S., Ohta, T., Kobayashi, M., Hanazato, T., and Yamagata, H. (1997). Gene 189, 73–77.
Vinogradov, S. N. Waltz, D. A., Pohajdak, B., Moens, L., Kapp, O. H., Suzuki, T., and Trotman, C.N.A. (1993). Comp.Biochem.Physiol[ B] 106, 1–26.
Urich, K. (1990). Comparative Animal Biochemistry, Springer, Berlin.
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Rosado-Ruiz, T., Antommattei-Pérez, F.M., Cadilla, C.L. et al. Expression and Purification of Recombinant Hemoglobin I from Lucina pectinata. J Protein Chem 20, 311–315 (2001). https://doi.org/10.1023/A:1010901701841
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DOI: https://doi.org/10.1023/A:1010901701841