Expression, purification and spectra characterization of neuroglobin
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.
Available from: Arnt J Raae
- "Reduced Ngb shows two weak peaks at 528 and 558 nm in addition to the strong absorption peak at 424 nm. These are typical absorption peaks shown in neuroglobin and also other globins   . Fig. 3 "
[Show abstract] [Hide abstract]
ABSTRACT: Neuroglobin (NGB) is a newly discovered member of the hemoglobin superfamily that is primarily expressed in the brain of humans
and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB
were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix
form when its concentration is less than 10 μmol/L. However, its α-helix content decreaes with the increase of concentration
in the range of 10–40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs
form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol
is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary
structure in either acidic or alkaline solution to some extent. Although increased temperature destabilizes the α-helices
of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.
Chinese Science Bulletin 10/2006; 51(21):2581-2585. DOI:10.1007/s11434-006-2144-7 · 1.58 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: pH-induced unfolding and refolding of apo-neuroglobin (apo-Ngb) were investigated by UV, fluorescence, circular dichroism (CD) spectra and light scattering measurements. Results revealed that apo-Ngb became partially unfolded at around pH 5.0, with evidences from a red shift in the fluorescence spectra, a decrease in the far-UV CD and a sharp peak in the light scattering intensity. Further lowering of the pH reversed these effects, suggesting that apo-Ngb folds back to a compact state. At pH 2.0, the apo-Ngb forms a folding intermediate known as molten globule (MG), which is possessed of native-like secondary structure and almost complete loss of tertiary structure. Based on these results, the acid-induced denaturation pathway of apo-Ngb can be illustrated from the native state (N), via a partially unfolded state (U(A)) to the molten globule state (MG).
Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 02/2010; 75(5):1600-4. DOI:10.1016/j.saa.2010.02.025 · 2.35 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.