Abstract
The expression, purification and spectra characterization of recombinant human neuroglobin (NGB) are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 (DE3) plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red soluble protein was obtained which showed a single band in electrophoresis. Electrospray ionization mass spectrometry (ESI-MS) showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of porphyrin, respectively, and the oxidized NGB has a strong absorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maximal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical a helix, and has a positive peak at 410 nm induced by heme. The NGB protein is stable when the pH is higher than 4.
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Burmester, T., Weich, B., Reinhardt, S. et al., A vertebrate globin expressed in the brain, Nature, 2000, 407(6803): 520–523.
Bashford, D., Chothia, C., Lesk, A. M., Determinants of a protein fold: unique features of the globin amino acid sequences, J. Mol. Biol., 1987, 196(1): 199–216.
Burmester, T., Ebner, B., Weich, B. S. et al., A novel globin type ubiquitously expressed in vertebrate tissues, Mol. Biol. Evol., 2002, 19(4): 416–421.
Awenius, C., Hankeln, T., Burmester, T., Neuroglobins from the zebrafish Danio rerio and the pufferfish Tetraodon nigroviridis, Biochem. Biophys. Res. Commun., 2001, 287(2): 418–421.
Zhang, C., Wang, C., Deng, M. et al., Full-length cDNA cloning of human neuroglobin and tissue expression of rat neuroglobin, Biochem. Biophys. Res. Commun., 2002, 290(5): 1411–1419.
Pesce, A., Bolognesi, M., Bocedi, A. et al., Neuroglobin and cytoglobin—Fresh blood for the vertebrate globin family, EMBO Rep., 2002, 3(12): 1146–1151.
Sun, Y., Jin, K., Peel, A. et al., Neuroglobin protects the brain from experimental stroke in vivo, Proc. Natl. Acad. Sci. USA., 2003, 100 (6): 3497–3500.
Sun, Y., Jin, K., Mao, X. O., Zhu, Y. et al., Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury, Proc. Natl. Acad. Sci. USA, 2001, 98(26): 15306–15311.
Sowa, A. W., Guy, P. A., Sowa, S. et al., Nonsymbiotic haemoglobins in plants, Acta. Biochim. Pol., 1999, 46(2): 431–445.
Hargrove, M. S., Brucker, E. A., Stec, B. et al., Crystal structure of a nonsymbiotic plant hemoglobin, Structure. Fold. Des., 2000, 8(9): 1005–1014.
Kriegl, J. M., Bhattacharyya, A. J., Nienhaus, K. et al., Ligand binding and protein dynamics in neuroglobin, Proc. Natl. Acad. Sci. USA, 2002, 99(12): 7992–7997.
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Clone: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 16–34.
Dewilde, S., Kiger, L., Burmester, T. et al., Biochemical characterization and ligand binding properties of neuroglobin, a novel member of the globin family, J. Biol. Chem., 2001, 276 (42): 38949–38955.
Djemel, H., Laurent, K., Sylvia, D., The redox state of the cell regulates the ligand binding affinity of human neuroglobin and cytoglobin, J. Biol. Chem., 2003, 278 (51): 51713–51721.
Wang, K., Bioinorganic Chemistry (in Chinese), Beijing: Tsinghua University Press, 1998, 120–121.
Strittmatter, P., The Nature of the Heme Binding in Microsomal Cytochrome b5, J. Biol. Chem., 1960, 235(8): 2492–2497.
Melo, E. P., Costa, S. M. B., Cabrai, J. M. S., Denaturation of a recombinant cutinase from Fuearium Solani in AOT-iso-octane reverse micelles:a steady-state fluorescence study, Photochem Photobiol., 1996, 63(1): 169–175.
Eaton, W. A., Hofrichter, J., Polarized absorption and linear dichroism spectroscopy of hemoglobin, Meth. Enzymol., 1981. 76(1): 175–226.
Sugiyama, T, Miura, R., Yamano, T. et al., A reversible spin conversion of cytochrome b5 at high temperatures, Biochem. Biophys. Res. Commun., 1980, 97(1): 22–27.
Tapan, K. D., Shyamalava, M., Samaresh, M., Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding, Eur. J. Biochem., 1998, 254(3): 662–670.
Msako, N., Yoshiki, S., Yoshimasa, Y., Circular dichroism of hemoglobin and its subunits in the Soret region, J. Biol. Chem., 1969, 244(6): 1651–1653.
Alessandra, P., Sylvia, D., Marco, N. et al., Human brain neuroglobin structure reveals a distinct mode of controlling oxygen affinity, Structure, 2003, 11 (9): 1087–1095.
Alessandra, P., Sylvia, D., Marco, N. et al., The human brain hexacoordinated neuroglobin three-dimensional structure, Micron., 2004, 35(1–2): 63–65.
De Young, L. R., Dill, K. A., Fink, A. L., Aggregation and denaturation of apomyoglobin in aqueous urea solution, Biochemistry, 1993, 32 (15): 3877–3886.
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Li, L., Ji, H., Zhao, C. et al. Expression, purification and spectra characterization of neuroglobin. Chin.Sci.Bull. 50, 1708–1713 (2005). https://doi.org/10.1360/982004-618
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DOI: https://doi.org/10.1360/982004-618