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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 Hip rep 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. U V-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 a bands of porphyrin, respectively, and the oxidized NGB has a strong absorption peak at 413 nm which corresponds to the transition of it 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. [ABSTRACT FROM AUTHOR]

Sensitive and accurate detection of DNA methyltransferase (MTase) is conducive to the understanding of the fundamental biological processes related to DNA methylation, clinical disease diagnosis, and drug discovery. Herein, a new fluorescence transducer based on Förster resonance energy transfer (FRET) between the donor upconversion nanoparticles (UCNPs) and the efficient acceptor gold nanorods (AuNRs) for MTase activity analysis and its inhibitor screening is presented. A double-strand DNA linker between UCNPs and AuNRs could be digested by restriction endonuclease HhaI, preventing the FRET process and recovering the upconversion luminescence (UCL) intensity. With the treatment of MTase, the cutting site was disturbed by the methylation of cytosine, blocking the enzyme digestion. The transducer presented here showed an excellent analytical performance toward MTase M.HhaI in the concentration range 0.08~24 U mL−1 with a detection limit of 0.057 U mL−1 calculated according to the UCL intensity changes at 656 nm excited by 980 nm CW laser, which is superior to most of the reported methods. Furthermore, the as-fabricated transducer also demonstrated high testing and screening capability toward enzyme inhibitors' evaluation. The method takes the advantage of low background fluorescence of UCNPs to improve the accuracy of the measurement, which can be developed as a general strategy for the analysis of various disease-related methyltransferase activity and their corresponding inhibitors, offering a promising strategy for high-performance diagnosis, high-efficient drug exploitation, and treatment effectiveness evaluation. [ABSTRACT FROM AUTHOR]

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 decreases 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. [ABSTRACT FROM AUTHOR]