Purification of recombinant proteins by chemical removal of the affinity tag

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.     

Structural characterization and electronic properties determination by high-field and high-frequency EPR of a series of five-coordinated Mn(II) complexes

The isolation, structural characterization, and electronic properties of a series of high-spin mononuclear five-coordinated Mn(II) complexes, [Mn(terpy)(X)(2)] (terpy = 2, 2':6',2' '-terpyridine; X = I(-) (1), Br(-) (2), Cl(-) (3), or SCN(-) (4)), are reported. The X-ray structures of the complexes reveal that the manganese ion lies in the center of a distorted trigonal bipyramid for complexes 1, 2, and 4, while complex 3 is better described as a distorted square pyramid. The electronic properties of 1-4 were investigated by high-field and high-frequency EPR spectroscopy (HF-EPR) performed between 5 and 30 K. The powder HF-EPR spectra have been recorded in high-field-limit conditions (95-285 GHz) (D < gbetaB). The spectra are thus simplified, allowing an easy interpretation of the experimental data and an accurate determination of the spin Hamiltonian parameters. The magnitude of D varies between 0.26 and 1.00 cm(-)(1) with the nature of the anionic ligand. Thanks to low-temperature EPR experiments, the sign of D was unambiguously determined. D is positive for the iodo and bromo complexes and negative for the chloro and thiocyano ones. A structural correlation is proposed. Each complex is characterized by a significant rhombicity with E/D values between 0.17 and 0.29, reflecting the distorted geometry observed around the manganese. Finally, we compared the spin Hamiltonian parameters of our five-coordinated complexes and those previously reported for other analogous series of dihalo four- and six-coordinated complexes. The effect of the coordination number and of the geometry of the Mn(II) complexes on the spin Hamiltonian parameters is discussed.     

Zinc(II) and cadmium(II) complexes containing the 2-{2-[3-(triethoxysilyl)-propylthio]ethylamino}ethylamino ligand

The new 2-{2-[3-(triethoxysilyl)propylthio]ethylamino}ethylamino SNN ligand, has been synthesized and fully characterized. Its donor properties towards zinc(II) and cadmium(II) have been investigated in order to simulate the metal uptake behavior in environmental applications. It reacts with ZnX₂ (X = Cl, Br and I) and CdCl₂ to form monomeric molecular complexes, MX₂(SNN)₂. Mass, i.r., ¹H- and ¹³C{¹H}-n.m.r. spectroscopies and elemental analyses reveal that, in these complexes, the metal attains its highest coordination number by linking to two nitrogen atoms of the ethylenediamine portion, and to two halogen atoms. The SNN ligand thus behaves as a bidentate four electrons donor, the thioether sulfur atom still remaining available for further coordination.     

Studies of mixed silica/alumina and silica/boric oxide materials

Multinuclear low temperature solution NMR and FTIR has been used to monitor the hydrolysis and polymerisation chemistry involved in the preparation of multicomponent silica/alumina and silica/boric oxide monolithic gels. Pre-hydrolysis of the silicon component (tetraethylorthosilicate) in the presence of low levels of water is an important factor in obtaining transparent materials. In order to obtain high homogeneity and minimise the subsequent precipitation of the fastest hydrolysing component, ethylacetonate (etac) has been used to modify the aluminum alkoxides. Solid state nmr and FTIR studies show that the borosilicate system contains Si-O-B bonds during the early stages of polymerisation but they are absent on final gelation. Thermal treatment to around 500°C is required to generate mixed Si-O-B bonds. Length of prehydrolysis has little affect on the nature of the Si-O-B gel materials but has a significant effect on the chemical nature of Si-O-Al materials. Longer silicon alkoxide prehydrolysis times lead to better defined materials.formerly Carole C. Perry.formerly at Chemistry Department, Brunel University, Uxbridge, Middlesex. UB8 3PH UK.     

Porphyrin architectures tailored for studies of molecular information storage

A molecular approach to information storage employs redox-active molecules tethered to an electroactive surface. Zinc porphyrins tethered to Au(111) or Si(100) provide a benchmark for studies of information storage. Three sets of porphyrins have been synthesized for studies of the interplay of molecular design and charge-storage properties: (1) A set of porphyrins is described for probing the effect of surface attachment atom on electron-transfer kinetics. Each porphyrin bears a meso-CH2X group for surface attachment where X = OH, SAc, or SeAc. (2) A set of porphyrins is described for studying the effect of surface-charge density in monolayers. Each porphyrin bears a benzyl alcohol for surface attachment and three nonlinking meso substituents of a controlled degree of bulkiness. (3) A set of porphyrins is described that enables investigation of on-chip patterning of the electrolyte. Each porphyrin bears a formyl group distal to the surface attachment group for subsequent derivatization with a molecular entity that comprises the electrolyte. Taken together, this collection of molecules enables a variety of studies to elucidate design issues in molecular-based information storage.     

PRODUCTS OF CHLOROPHYLL PHOTODEGRADATION–2. STRUCTURAL IDENTIFICATION

Abstract— Hydrophilic products of chlorophyll a photodegradation are structurally identified using UV/visible and fourier transform infrared spectrophotometry and gas chromatography-mass spectrometry. The major peak detected during reverse-phase high performance liquid chromatography of the colourless photodegradation products is identified as glycerol. Lactic, citric, succinic and malonic acids, as well as alanine, are also identified.     

Interference-free biosensor based on screen-printing technology and sol-gel immobilization for determination of acetaldehyde in wine

A monoenzymatic amperometric biosensor was developed for the detection of acetaldehyde. The sensor is based on the association of screen-printed carbon electrodes and aldehyde dehydrogenase immobilized by a sol-gel entrapment method. Modification of screen-printed carbon electrodes with Reinecke salt of Meldola's Blue (MBRS) resulted in highly sensitive and interference-free nicotinamide-adenine dinucleotide (NADH) detectors. Based on MBRS-mediated oxidation of NADH at -150 mV versus pseudo Ag/AgCl, acetaldehyde was determined in the range 10-260 microM, compatible with wine quality monitoring. The method of immobilization based on sol-gel entrapment was optimized to obtain the best compromise between sensitivity and operational stability. The sensor response was stable for 40 consecutive assays with methyltrimethoxysilane used as alkoxide precursor, thus allowing a possible calibration of the sensor before each measurement. The biosensors were used to analyze French wines. The method was validated with a commercially available enzymatic kit based on a standard spectrophotometric method.