Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

Liquid–liquid extraction of a recombinant protein with reverse micelles

Recombinant cytochrome b₅ was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b₅ to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1–2 mol dm^?3 NaCl) and low temperatures (4°C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.     

The fracture stress of chemical vapour deposited diamond

The factors which control the fracture stress of chemical vapour deposited diamond have been studied using the 3-point bend geometry. Fracture stress values of 300–800 MPa for the growth side and 600–1200 MPa for the nucleation side were recorded for samples of thickness 0.4–2.4 mm. A Weibull modulus of 23 was calculated for the growth surface data, showing that the fracture stress variability was low for a brittle material. A theory based on these results demonstrates that the material behaviour is remarkably simple, depending only on the grain size and the sample thickness, regardless of wide variations in other parameters such as optical transmission and stress state. The paper also contains a possible explanation for this well-defined behaviour based on microstress variations resulting from differences in defect density in different growth sectors within a grain.     

Superconductivity at differentTc in CdBa2Ca2Cu3Oy

A Cd analogue of the Tl and Hgn =3 series with nominal composition CdBa₂Ca₂Cu₃O_y has been synthesized. The samples were superconducting according to magnetic susceptibility measurements. The critical temperature was 103 or 107 K depending on the preparation conditions. The EDX analysis revealed the presence of Cd-1111, Cd-1121, and Cd-2333 as minor phases. The observed diamagnetic effects were attributed to the differentT _c of these phases.     

锆(Ⅳ)-3,5-diBr-PADAP-安替比林三元配合物的分光光度研究与应用

本文提出在安替比林存在下,以3,5-diBr-PADAP为显色剂,分光光度测定微量锆的新方法。在pH1～2的HCl介质中,锆(W)与试剂和安替比林形成配合比为1:1:1的红色三元配合物,最大吸收值在615nm波长处,摩尔吸光系数ε=1.35×10~5。锆(Ⅳ)量在0～25μg/25ml范围内,遵守比尔定律。方法简单、快速、准确。若采用钽试剂—苯萃取法分离共存离子,可适用于钽铌矿和合金中痕量锆的测定。     

Phase composition and properties of solid solutions of GdFeO3–GdInO3 bulks

Solid solutions of the GdFeO₃–GdInO₃ system were prepared at 1550 °C by ceramic powder processing. The formulated composition was Gd(Fe_1−xIn_x)O₃ (GFI) with the indium contents at x = 0, 0.25, 0.5, 0.75, and 1.0. A stable phase of Gd(Fe_1/3In_2/3)O₃ in our system was identified by X-ray diffraction and phase composition analysis. Multi-phase morphologies were observed for GFI bulks with x = 0.5 and 0.75. Dielectric and electrical properties of the GFI bulks were investigated. The addition of 25% In³⁺ in GdFeO₃ had an obvious enhancement in polarization and led to an elevated resonance frequency. Dielectric properties of GFI bulks except GdInO₃ were strongly dependent upon the test frequency, which corresponded to the response of polarization mechanism. GdInO₃ displayed as a stable dielectric, which was frequency- and temperature-insensitive. GdInO₃ was thermally activated and became leaky until above 600 °C.     

Photocatalytic degradation of 2,2‐bis(4‐hydroxy‐3‐methylphenyl) propane (BPP) based on molecular recognition interaction

BACKGROUND: Endocrine disruptors in the aquatic environment and their potential adverse effects are currently issues of concern. One of these endocrine disruptors is 2,2‐bis(4‐hydroxy‐3‐methylphenyl)propane (BPP). In this work the molecular recognition interaction of BPP with β‐cyclodextrin (β‐CD) was studied using IR spectroscopy and steady state fluorescence spectroscopy, and the photocatalytic degradation behaviour of BPP based on molecular recognition interaction was investigated in a TiO₂/UV–visible (λ_max = 365 nm) system. This might provide a new method for the treatment of some organic pollutants in wastewater. RESULTS: β‐CD reacts with BPP to form a 1:1 inclusion complex, the formation constant of which is 4.94 × 10³ L mol^?1. The photodegradation rate constant of BPP after molecular recognition by β‐CD showed a 1.42‐fold increase in the TiO₂/UV–visible (λ_max = 365 nm) system. The photodegradation of BPP depended on the concentration of β‐CD, the pH value, the gaseous medium and the initial concentration of BPP. The photodegradation efficiency of BPP with molecular recognition was higher than that without molecular recognition. After 100 min of irradiation the mineralisation efficiency of BPP after molecular recognition by β‐CD reached 94.8%, whereas the mineralisation efficiency of BPP before molecular recognition by β‐CD was only 40.6%. CONCLUSION: The photocatalytic degradation of BPP after molecular recognition by β‐CD can be enhanced in the TiO₂/UV‐visible (λ_max = 365 nm) system. This enhancement is dependent on the enhancement of the adsorption of BPP, the moderate inclusion depth of BPP in the β‐CD cavity and the increase in the frontier electron density of BPP after molecular recognition. Copyright © 2008 Society of Chemical Industry     

One‐step production of 2,3‐butanediol from starch by secretory over‐expression of amylase in Klebsiella pneumoniae

BACKGROUND: 2,3‐Butanediol (2,3‐BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3‐BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3‐BD directly from starch. RESULTS: In this work, gene malS coding for α‐amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over‐expression of α‐amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one‐step production of 2,3‐BD from starch was carried out by engineered K. pneumoniae. A 2,3‐BD concentration of 3.8 g L^?1 and yield of 0.19 g 2,3‐BD g^?1 starch were obtained after 24 h fermentation. CONCLUSION: The one‐step production of 2,3‐BD from starch was achieved by secretory over‐expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. Copyright © 2008 Society of Chemical Industry     

Material removal and surface damage in EDM of Ti3SiC2 ceramic

Material removal and surface damage of Ti₃SiC₂ ceramic during electrical discharge machining (EDM) were investigated. Melting and decomposition were found to be the main material removal mechanisms during the machining process. Material removal rate was enhanced acceleratively with increasing discharge current, i_e, working voltage, u_i, but increased deceleratively with pulse duration, t_e. Microcracks in the surface and loose grains in the subsurface resulted from thermal shock were confirmed, and the surface damage in Ti₃SiC₂ ceramic led to a degradation of both strength and reliability.     

Application of artificial neural networks to the analysis of two‐dimensional fluorescence spectra in recombinant E coli fermentation processes

A two‐dimensional (2D) spectrofluorometer was used to monitor various fermentation processes with recombinant E coli for the production of 5‐aminolevulinic acid (ALA). The whole fluorescence spectral data obtained during a process were analyzed using artificial neural networks, ie self‐organizing map (SOM) and feedforward backpropagation neural network (BPNN). The SOM‐based classification of the whole spectral data has made it possible to qualitatively associate some process parameters with the normalized weights and variances, and to select some useful combinations of excitation and emission wavelengths. Based on the classified fluorescence spectra a supervised BPNN algorithm was used to predict some of the process parameters. It was also shown that the BPNN models could elucidate some sections of the process's performance, eg forecasting the process's performance. Copyright © 2005 Society of Chemical Industry