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Expression, purification and spectra characterization of neuroglobin

Chinese Science Bulletin (Impact Factor: 1.37). 50(16):1708-1713. DOI: 10.1360/982004-618

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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