The expression, purification and spectra char-acterization 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 ...The expression, purification and spectra char-acterization 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 solu-ble protein was obtained which showed a single band in elec-trophoresis. 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 por-phyrin, respectively, and the oxidized NGB has a strong ab-sorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maxi-mal 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 α helix, and has a positive peak at 410 nm induced by heme. The NGB protein is stable when the pH is higher than 4.展开更多
文摘The expression, purification and spectra char-acterization 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 solu-ble protein was obtained which showed a single band in elec-trophoresis. 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 por-phyrin, respectively, and the oxidized NGB has a strong ab-sorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maxi-mal 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 α helix, and has a positive peak at 410 nm induced by heme. The NGB protein is stable when the pH is higher than 4.
