Chemo-enzymatic synthesis of arginine-based gemini surfactants

A novel chemo-enzymatic synthesis of arginine-based gemini cationic surfactants bis(Args) is reported. These compounds consist of two single N(alpha)-acyl-arginine structures connected through the alfa-carboxylic groups of the arginine residues by a alpha, omega-diaminoalkane spacer chain. N(alpha)-Acyl-L-arginine alkyl ester derivatives were the starting building blocks for the synthesis. The best strategy found consisted of two steps. First, the quantitative acylation of one amino group of the spacer by the carboxylic ester of the N(alpha)-acyl-arginine took place spontaneously, at the melting point of the alpha,omega-diaminoalkane, in a solvent-free system. The second step was the papain-catalyzed reaction between another N(alpha)-acyl-arginine alkyl ester and the free aliphatic amino group of the derivative formed in the first step. Reactions were carried out in solid-to-solid and solution systems using low-toxic potential solvents. Changes in reaction performance and product yield were studied for the following variables: organic solvent, support for enzyme deposition and substrate concentration. The best yields (70%) were achieved in solid-to-solid systems and in ethanol at a(w) = 0.07. Bis(Args) analogs of 8, 10 and 12 carbon atoms using 1,3-diaminopropane and 1, 3-diamino-2-hydroxy-propane as hydrocarbon spacers were prepared at the 6-7 gram level employing the methodology developed. The overall yields which include reaction and purification varied from 51% to 65% of pure (97-98% by HPLC) product.     

Sequestration of bacterial lipopolysaccharide by bis(Args) gemini compounds

Gemini compounds of the type N(alpha),N(omega)-bis(N(alpha)-lauroyl arginine)alpha,omega-alkylenediamides or bis(Args) bind bacterial lipopolysaccharide and neutralize endotoxic activity in in vitro tumor necrosis factor-alpha and nitric oxide release assays. Sequestration of lipopolysaccharide results in protection in a murine model of endotoxemia. However, the bis(Args) compounds are cytotoxic by virtue of being highly membrane-active. The development of less surface-active analogues may yield potentially therapeutically useful compounds for the treatment of Gram-negative sepsis.     

离子交换层析纯化透明质酸

考察6种离子交换树脂的静态吸附解析效果,选出201＊7阴离子交换树脂填柱,确定洗脱流速为0.6mL/min,40mL0.3mol/LNaCl和50mL0.5mol/L NaCl双浓度洗脱,实现透明质酸和杂蛋白的分离。制得透明质酸产品蛋白含量为0.057%,葡萄糖醛酸含量为43%,平均相对分子质量大于1.1×10^6,收率为54%,符合医用级透明质酸行业标准的要求。     

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography

Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.     

Purification of hormone-sensitive lipase by high-performance ion exchange chromatography

We have developed an improved method for purification of hormone-sensitive lipase from adipose tissue. The method employs two preparative high-performance ion-exchange chromatography steps on Mono Q and Mono S after detergent solubilization and partial fractionation of the enzyme by gradient sievorptive chromatography on QAE-Sephadex. About 0.2 mg of greater than 70% pure enzyme is prepared at 10% yield within 6-7 days from adipose tissue of 500 rats. This protocol is a major improvement over the previously established procedure in terms of accessibility, rapidity, enzyme purity, and yield.     

Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography

A purification procedure for type E botulinum neurotoxin has been developed, based solely on high-performance ion exchange chromatography. The method exploits the differential chromatographic behavior of the free neurotoxin versus the neurotoxin-protein 12S complex. The high purity of the product was demonstrated with sodium dodecyl sulfate-gel electrophoresis and amino acid sequencing. Beginning with dialyzed crude extract, at least 4 mg of pure neurotoxin could be obtained in two working days. The method has been adapted to user-prepared columns for processing large volumes of crude neurotoxin in one batch.     

Purification of glucagon by subunit exchange chromatography

Glucagon was immobilized onto Sepharose matrices activated with CNBr or tresyl chloride, as a function of several parameters including pH of coupling, concentration of added polypeptide, and presence or absence of urea. The hormone was linked to the matrix through a single point per molecule, namely, the epsilon -amino group of Lys(12) when the coupling was carried out at alkaline pH, or the imidazole group of His(1) when the coupling was carried out at acidic pH. Glucagon immobilized at alkaline pH interacted specifically with soluble glucogon. The extent of self-association was similar to that of free glucagon, which exists in solution in a monomer-trimer equilibrium whose association constant is highly dependent on the characteristics of the buffer (pH, ionic strength, and nature of anions). The immobilized hormone proved to be suitable for the purification of the free one from a pancreatic extract. After a preliminary treatment with charcoal-dextran, the extract was percolated on a glucagon-Sepharose column under associating conditions (high concentrations of salting out anions and alkaline pH) and then, following a washing to remove extraneous compounds, the specifically bound hormone was eluted under dissociating conditions (low ionic strength). The subunit exchange chromatography of the extract gave a ca. 90% pure product. The overall recovery of the process was ca. 66%. The leakage of immobilized hormone was 40% in the case of CNBr activation of Sepharose and 15% in the case of tresyl chloride activation, after an eight-day treatment under working conditions.     

离子交换色谱法对大肠杆菌表达的人重组骨形态发生蛋白-7的纯化

重组大肠杆菌高量表达重组人骨形态发生蛋白-7(rhBMP-7),每升培养液约得到湿菌体3g,其中目的蛋白约占菌体总蛋白量的40％。裂解离心,用低浓度变性剂洗涤初步纯化包涵体,上清中无目的蛋白损失;将包涵体溶解于高浓度变性剂溶液中,目的蛋白纯度提高到60％;然后在不同条件下用离子交换色谱法对变性状态下的蛋白质进行纯化,绝大部分杂蛋白被除去,目的蛋白纯度达95％以上;改变条件,可以减少rhBMP-7损失;用Western blot对目的蛋白进行特异性鉴定。     

Purification of chicken testicular müllerian inhibiting substance by ion exchange and high-performance liquid chromatography

A highly purified protein molecule was obtained from the secretory proteins of 8-week-old chicken testes using ion-exchange column chromatographic procedures, including DEAE Bio-Gel A, CM Bio-Gel A, wheat germ lectin columns, and high-performance liquid chromatographic (hplc) separation techniques. This protein molecule has a molecular weight of 74,000 Da (74K protein). The isolated 74K protein induces regression of chicken Müllerian ducts grown in vitro. The 74K protein does not cause regression of cultured embryonic intestine or Wolffian duct. When the total testicular secretory proteins are resolved in a two-dimensional gel electrophoresis, approximately 120 polypeptides are obtained. The purified 74K protein has a pI of 6.1. Analysis of amino acid composition indicates that the 74K protein is relatively acidic in nature with a ratio of acidic to basic amino acids of 1.93.     

Bovine plasma protein fractionation by ion exchange chromatography

An ion exchange chromatography process was developed to separate the main protein fractions of bovine blood plasma using a composite material, Q-HyperD resin, and a gel material, DEAE-Sepharose. The experiments were carried out at semipreparative scale. It was necessary to establish analytical methods of electrophoresis and HPLC to identify the fractionated proteins. Results show that these materials are able to adequately fractionate different protein groups from the raw blood plasma. This method may be used to avoid chemical fractionation using agents such as ethanol or PEG and, thus, decrease protein denaturation of the different fractions to be used for research or pharmaceutical purposes. The Q-HyperD resin presents a better retention capacity for plasma protein than DEAE-Sepharose under the experimental conditions employed.     

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.     

Matrix assisted refolding of proteins by ion exchange chromatography

Two different approaches of matrix assisted refolding have been evaluated and compared to conventional refolding by dilution. Bovine alpha-lactalbumin was used for the studies as model protein. It was adsorbed under denaturing conditions on an ion exchange matrix and refolding was completed on the column prior to elution or, depending on the buffer system, in the eluate. Agarose based chromatography matrices showed high capacities for the denatured alpha-lactalbumin. A positive effect on the yield of refolded protein by the matrix could be observed for Fractogel EMD DEAE and a negative for Toyopearl DEAE 650M, DEAE Sepharose FF and Q Sepharose FF. In the case of Fractogel EMD DEAE the ion exchange surface might act as a folding helper. This property may be caused by the grafted polymers. For Source 30Q only a marginal negative influence on the refolding kinetics was observed, thus the ion exchanger is only a mean for removal of chaotropic agents. Refolding on the column is characterized by a low yield but high productivity due to significant reduction of refolding time.     

Kinetic studies of clavulanic acid recovery by ion exchange chromatography

Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus. Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid–liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 °C and 20 °C during adsorption and 30 °C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations. This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of lactic acid adsorption by ion exchange chromatography

Background

The polyacrylic resin Amberlite IRA-67 is a promising adsorbent for lactic acid extraction from aqueous solution, but little systematic research has been devoted to the separation efficiency of lactic acid under different operating conditions.

Methodology/Principal Findings

In this paper, we investigated the effects of temperature, resin dose and lactic acid loading concentration on the adsorption of lactic acid by Amberlite IRA-67 in batch kinetic experiments. The obtained kinetic data followed the pseudo-second order model well and both the equilibrium and ultimate adsorption slightly decreased with the increase of the temperature at 293–323K and 42.5 g/liter lactic acid loading concentration. The adsorption was a chemically heterogeneous process with a mean free energy value of 12.18 kJ/mol. According to the Boyd^_plot, the lactic acid uptake process was primarily found to be an intraparticle diffusion at a lower concentration (<50 g/liter) but a film diffusion at a higher concentration (>70 g/liter). The values of effective diffusion coefficient D_i increased with temperature. By using our Equation (21), the negative values of ΔG° and ΔH° revealed that the adsorption process was spontaneous and exothermic. Moreover, the negative value of ΔS° reflected the decrease of solid-liquid interface randomness at the solid-liquid interface when adsorbing lactic acid on IRA-67.

Conclusions/Significance

With the weakly basic resin IRA-67, in situ product removal of lactic acid can be accomplished especially from an open and thermophilic fermentation system without sterilization.