Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system.

AIM: To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target. METHODS: α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis. RESULTS: Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel. CONCLUSION: We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.

High yield and efficient expression and purification of the human 5-HT3A receptor.

AIM: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. METHODS: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). RESULTS: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. CONCLUSION: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor.

[Study on the method for rapid determination of oil concentration in water].

The rapid determination of oil concentration in water was investigated with three dimensional fluorescence spectra and parallel factor analysis (PARAFAC), and the influence of dissolved organic matter on the measurement of oil concentration was analyzed mainly. No. 0 diesel oil and humic acid was used for measuring sample in the experiment, the fast separation of No. 0 diesel oil fluorescence spectra from the overlapped spectrum of humic acid was studied through changing humic acid concentration, the contents of No. 0 diesel oil was obtained under different humic acid simulation conditions, the maximum average error was 1.67%, and the relative standard deviation was less than 1.29%. The results showed the possibility of oil concentration fast determination with a variation of dissolved organic matter in water based on three dimensional fluorescence spectra technique and PARAFAC.

[Analysis of the characters of organic matter in water using spectral fluorescence signature and fitting Gaussian].

A sort of analytical method of fast diagnosis of organic matter in water is discussed. The total luminescence spectra (TLS) of water samples in combination with laser induced fluorescence (LIF) measurements using 532 nm wavelength excitation source were measured in the laboratory. The spectra of dissolved organic matter (DOM) and chlorophyll a (Chl a) were pointed out and obtained with spectral fluorescence signature (SFS) technique. The spectrum of water Raman scattering and fluorescence of DOM and Chl a were separated from TLS with fiting Gaussian of the least squares method, and a high correlation coefficient excelling 0.996 4 was obtained. The results indicated the possibilities of water quality monitoring in real time and on line based upon SFS technique and fitting Gaussian of the least squares method.

[The influence of temperature change on determined concentrations of dissolved organic matter in water].

The influence of temperature change on the determined concentrations of dissolved organic matter (DOM) in water was investigated by laser induced fluorescence (LIF) technique in laboratory. The temperature was altered within the range of 20 to 75 degrees C, and the intensity of DOM fluorescence and water Raman scattering were found to decrease with rising temperature. A linear fit of the normalized fluorescence intensity versus temperature yielded a mean value of the temperature coefficient of -5.24 x 10(-4) x degrees C(-1), and a mean value of the temperature coefficient of -3.42 x 10(-3)(mg x L(-1)) x degrees C(-1) was obtained according to the relationship between normalized fluorescence intensity and concentration of DOM. The temperature change will cause relative changes of up to 8.45% in DOM concentration within the range of 20 to 75 degrees C if we assume that the normalized fluorescence intensity remains the same.

[An analysis of quenching characteristics of DOM in water by laser induced fluorescence].

In the experiment an excitation resource at 355 nm from a third harmonic Q-switched Nd:YAG laser was used, and the commercial humic acid of different concentrations was used as the research samples. The fluorescence spectrum of dissolved organic matter (DOM) in combination with laser-induced fluorescence (LIF) measurements was measured in the laboratory, and the characteristics of fluorescence quenching of DOM were analyzed. The results show that the intensity of water Raman scattering and DOM fluorescence was reduced gradually and increased linearly respectively with raising the concentration of humic acid. The water Raman scattering signal was absorbed almost completely by the ground state molecules of DOM at 40 mg x L(-1), and the fluorescence intensity of DOM reached a maximum at 16 mg x L(-1) and decreased slowly with further increasing the concentration of humic acid. Consequently, we can achieve better monitoring of DOM in water through analyzing the characteristics of fluorescence quenching at different concentrations of humic acid.

[The fluorescence emission of water at upconversion of frequency by laser induced fluorescence].

The fluorescence spectra of several types of water samples in combination with laser-induced fluorescence (LIF) measurements using 532 nm wavelength excitation source were measured in the laboratory, and the fluorescence matters such as dissolved organic matter (DOM), chlorophyll a (Chl a) and carotenoids were pointed out with spectral fluorescent signature (SFS) technique. It is considered that the peak at 455 nm in the spectrum is contributed by carotenoid (PPC). Finally, the dynamic model of PPC's fluorescence emission is showed.

[Study the characteristics of dissolved organic matter and turbidity of water using laser induced fluorescence and laser scattering].

In the experiment a frequency doubled radiation of Nd:YAG laser was used for excitation at 532 nm. The fluorescence spectra of dissolved organic matter (DOM) and chlorophyll a (Chl-a) in several types of water samples with laser-induced fluorescence (LIF) measurements using a 532 nm wavelength excitation source were measured in the laboratory, and the spectral characteristics of DOM and Chl-a were analysed. The characteristics of turbidity were investigated by measuring the amount of scattering light of suspended particles in the water volume. The curve of scattering intensity against corresponding turbidity of SiO2 is showed. The possibilities of water quality monitoring based upon the fluorescence spectral characteristics of contaminations and the turbidity by means of LIF method.