Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.     

How to build optically activeα-amino acids

Summary Various methodologies published in the literature dealing with-amino carboxylic acid asymmetric synthesis are presented in a digest form. In each case, only some recent or most typical works are mentioned.     

A fluorimetric method for the determination of trace pentachlorophenol, based on its inhibitory effect on the redox reaction between the improved Fenton reagent and rhodamine B.

A sensitive fluorimetric method is presented and discussed for the determination of pentachlorophenol in aqueous solutions. This method is based on the inhibitory effect of pentachlorophenol on the reaction of conventional Fenton [Fe(III) + H(2)O(2)] reagent with rhodamine B in the medium of perchloric acid, which results in the fluorescence quenching of rhodamine B. It was further found that the sensitivity for the determination was improved significantly when the molecular ligand EDTA was added. This improved system was therefore presented for the determination of pentachlorophenol. The characteristics of the excitation and emission spectra, optimization of the experimental conditions, the stability of the system and the influence of foreign matter have all been investigated. Under optimal conditions, the linear range for the determination of pentachlorophenol is 12-480 ng/mL with a 3sigma limit of detection of 0.96 ng/mL. Compared with the conventional Fenton system, the improved system shows obvious advantages in both sensitivity and selectivity. By combination with the pretreatment of samples using ion exchange resins and XDA-1 absorption resin, the improved Fenton method was used for the first time for the determination of pentachlorophenol in synthetic samples and natural water samples, and satisfactory results, in agreement with those of the HPLC method, were achieved. The possible mechanism of the reactions has also been discussed. Copyright (c) 2007 John Wiley & Sons, Ltd.     

A novel cross-linking reagent for bovine hemoglobin modification

A novel cross-linking reagent, methoxypolyethelene glycol-glutamic acid, was synthesized and used to modify bovine hemoglobin. Bis-tetrameric hemoglobin with moderate affinity for O₂ was obtained under the controlled reaction conditions.     

Flavanol binding of nuclei from tree species

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cells radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.Abbreviations DMACA p-Dimethylaminocinnamaldehyde Communicated by W. Barz     

Porcine FAD-containing monooxygenase metabolizes lidocaine, bupivacaine and propranolol in vitro

Lidocaine, bupivacaine and propranolol are amines that can be expected to act as substrates for FAD-containing monooxygensae (FMO) (EC 1. 14. 13. 8). We found that FMO metabolizes lidocaine, bupivacaine and propranolol. The Km and Vmax values of lidocaine, bupivacaine and propranolol for FMO are 143, 408 and 210 microM, and 145, 119 and 135 nmol/min/mg FMO protein, respectively. The lipophilicity of the drugs decreased in the following order: lidocaine>propranolol>bupivacaine, under our experimental conditions. Furthermore, the metabolic products of FMO were separated by high-performance liquid chromatography and analyzed by gas chromatography-mass spectrometry, and were found to be the N-oxides and N-hydroxylamines of the respective drugs. These findings suggest that lidocaine, bupivacaine and propranolol are substrates for FMO, and the enzymatic toward lidocaine or bupivacaine may be inhibited exclusively and competitively by propranolol.     

悬浮培养法生产血型单抗的影响因素研究

目的：建立单克隆抗体血型定型试剂悬浮生产法最佳生产方案。方法：通过研究影响细胞生长及抗体产量的主要因素,找出合理的生产方法。结果：理论上用该方法每月可生产抗体１００万ｍｌ,比原贴壁培养法的产量提高数十倍。结论：建立一种简便、实用的血型定型试剂工业化大生产方法。     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms.

Some physiological factors which control the rate of induction of the Ca²⁺-induced membrane transition (the term “Ca²⁺-induced membrane transition” is defined in the introduction) have been systematically investigated. To exclude the complicating factors of electron flow and energization, the transition was studied in mitochondria depleted of endogenous substrate, in the presence of uncoupler. In these mitochondria the transition could be induced by Ca²⁺, whether entry was mediated by ruthenium red-sensitive permeation or by A23187 facilitated diffusion. The rate of the transition was reduced fivefold by any agent which caused complete reduction of endogenous NAD. The rate of the transition was increased threefold by the exchange of endogenous ADP for phosphoenolpyruvate. A further increase was found on the addition of atractyloside, but bongkrekic acid caused inhibition. Addition of uncoupler to energized mitochondria when the endogenous NAD was already fully oxidized caused a stimulation of the transition. From these observations we conclude that mitochondria have a set of protective mechanisms (the term “protective mechanism” refers to the means by which these agents inhibit the Ca²⁺-induced transition; such a mechanism could be through allosteric interactions between the sites of binding of inhibitor and Ca²⁺; it would, however, be premature to conclude this on the basis of this paper) involving endogenous NADH, ADP, and energization which regulate the rate of the Ca²⁺-induced transition. ADP appears to work at two sites: one site which is internal, and another at the ADP/ATP translocase. In addition, we conclude that the transition requires neither electron flow nor energy, but rather the mere accessibility of some internal site to Ca²⁺. Finally, the key roles played by the protective agents in metabolism give the cell great potential flexibility in regulating the Ca²⁺-induced transition. This degree of control suggests that the transition has substantial physiological significance.