Purification of the C1 repressor of bacteriophage P1 by fast protein liquid chromatography

A fast protein liquid chromatographic method is described for the purification of the C1 repressor of bacteriophage P1 and its truncated form C1*. By using one crude extract, both repressor proteins were purified in parallel to homogeneity and were shown to interact specifically with P1 operator DNA in vitro. The method involves an affinity chromatographic step on heparin-Sepharose, followed by a combination of ion-exchange chromatography on Q Sepharose and S Sepharose. The availability of a homogeneous preparation of the phage repressor is a prerequisite for studies on its structure-function relationship.     

Sepharose derivatives containing citric acid as affinity ligand. Purification of fumarase

Six Sepharose derivatives, in which citrate was immobilized via methylene carbons, were prepared by coupling of the alpha- and beta-isomers of citrylpolymethylenediamine to Sepharose. The purification of fumarase from pig heart was dependent on the length of the spacer arm, but not on the isomeric configuration of the immobilized citrate. Gels having six methylene carbons had the largest adsorption capacity for the enzyme and therefore were the most suitable for use in affinity columns for its purification. Affinity chromatography with these gels was followed by hydrophobic interaction chromatography on an octamethylenediamine-Sepharose column.     

Weak anion-exchange hypercrosslinked sorbent in on-line solid-phase extraction–liquid chromatography coupling to achieve automated determination with an effective clean-up

A mixed-mode polymeric sorbent was on-line coupled to liquid chromatography (LC) for the first time and applied to the selective solid-phase extract a group of pharmaceuticals in complex environmental water samples. The mixed-mode polymeric sorbent is a high-specific surface area hypercrosslinked polymer resin (HXLPP) in the form of monodisperse microspheres further modified with 1,2-ethylenediamine (EDA) moieties. These properties allow its application as a weak anion-exchange (WAX) sorbent in the on-line solid-phase extraction (SPE) coupling. The on-line SPE-LC method developed using the HXLPP-WAX sorbent was successfully applied to percolate a large volume of ultrapure (500 ml), river (250 ml) and effluent sewage (100 ml) water samples. In all the cases, the HXLPP-WAX resin provided near total recoveries of the most acidic compounds studied and clean chromatograms. This is because the ion-exchange interactions enable a washing step to be added to the SPE protocol that removes the compounds with weak acidic, neutral and basic properties from the sample matrix.     

Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.     

混合色谱填料的研究进展

随着科学技术的发展,人们需要分离分析的样品越来越复杂,尤其是多肽、蛋白质类生物样品的复杂性使得单一模式色谱难以满足分离分析的要求。混合模式色谱因其独特的分离性能,可以在一次分离中获得与多维色谱相当的分离效果,而且可以避免多维色谱系统结构复杂、流动相兼容性差、分析时间长等问题,成为近年来的研究热点之一。混合模式色谱的研究重点是色谱固定相的设计与开发。混合模式色谱固定相包括反相/离子交换混合固定相、反相/亲水混合固定相、亲水/离子交换混合固定相、两性离子交换混合固定相及三相混合固定相。本文综述了近年来混合模式色谱填料的研究及应用进展,并对混合模式色谱及固定相的发展前景进行了展望。     

A comparison of various commercially-available liquid chromatographic supports for immobilization of enzymes and immunoglobulins

A number of commercially-available, activated supports were evaluated and compared for the immobilization of enzymes (alkaline phosphatase, glucose oxidase and peroxidase) and human immunoglobulin G (IgG). The supports studied included pressure-stable, epoxy-activated acrylate-based supports (Separon HEMA 1000 and Eupergit C); agarosebased, epoxy-, cyanogen bromide-, glutaraldehyde- or N-hydroxysuccimide-activated supports (epoxy-activated or cyanogen bromide-activated Sepharose, ACT-Ultrogel AcA 22, Reacti-Gel 6X and Affi-Gel 10); and glass bead-based, activated supports (CDI- and NHS-Glycophase). As expected, the pH required for maximum protein immobilization and retention of activity varied with both protein and support. For example, the amount of alkaline phosphatase coupled was maximum at pH 3 or 5 for most supports, but retention of activity was greatest for immobilization at pH 7, 9 or 11. Glucose oxidase and peroxidase coupling and activity retention in general showed less variation in optimal coupling pH. Coupling of IgG and retention of anti-IgG binding activity were both optimal at a coupling pH of 9 or 11. The Separon HEMA-IgG support made in these studies was also utilized for rapid h.p.l.c, purification of anti-IgG from serum.     

Reinigung der D-Oxynitrilase aus Mandeln mit Hilfe der Affinitäts-Chromatographie

A Rapid Purification of D -Oxynitrilase from Almond Meal by Affinity Chromatography Oxynitrilase from almond meal is capable of catalyzing the stereospecific addition of cyanide to a variety of aldehydes. Thus, the enzyme is potentially useful in the synthesis of optically active cyanohydrins on a preparative scale [1]. As the currently available purification procedures for this enzyme [2] are rather tedious, we have elaborated a simple and rapid procedure based on affinity chromatography. An inhibitor for the enzyme, methyl p-(3-aminopropoxy)benzoate (4) , has been synthesized and attached covalently to Sepharose 4B as a solid matrix (5) . With this affinity gel it was possible to prepare the D -oxynitrilase in a simple procedure with high yields.     

混合模式色谱分离材料的研究及其应用进展

近年来混合模式色谱以其独特的分离特性受到人们越来越多的关注。混合模式色谱的种类主要集中在反相/离子交换混合模式色谱(reversed-phase/ion-exchange mixed-mode chromatography, RPLC/IEX),亲水作用/离子交换混合模式色谱(hydrophilic interaction/ion-exchange mixed-mode chromatography, HILIC/IEX),反相/亲水作用混合模式色谱(reversed-phase/hydrophilic interaction mixed-mode chromatography, RPLC/HILIC)等混合模式。两种或多种机理混合使用,往往在分离选择性和色谱峰形等方面能得到不同于单一模式操作所得到的效果,分离选择性以及色谱峰形等都能得到极大的改善与提高,这使得混合模式色谱渐渐进入研究者们的视野。混合模式色谱的研究多数集中在色谱填料的设计。混合模式色谱填料的应用主要针对生物样品的分离分析。该文综述了近年来混合模式色谱的研究及其应用进展,并展望了混合模式色谱的发展。     

Purification of the pregnancy zone protein by affinity chromatography.

A method for purification of the pregnancy zone protein (PZP) by affinity chromatography was developed. A monospecific immunoglobulin fraction, covalently coupled to Sepharose 4B, was used as binding agent and the elution conditions for PZP are described. The purified protein was shown to have identical properties compared to native PZP with regard to molecular weight, immunodiffusion precipitation and immunosuppressive activity.     

Ethylenediamine-Derived Chromatographic Ligand to Separate BSA,Lysozyme, and RNase A

Chromatography has become an essential tool for the purification of proteins, since most purification schemes involve some forms of this methodology. Recently, using chromatographic matrices prepared from symmetrical aminosquarylium cyanine dyes immobilized on Sepharose via a central alkylamino residue, we were able to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. Following this, we envisioned that the immobilization of an asymmetric squarylium dye bearing an N-carboxyethyl group in one of its ending nuclei, on ethylenediamine-activated Sepharose, through EDC/NHS amidation coupling, could enhance the ligand’s mobility and improve the interactions with the target proteins. The prepared support was found to separate an artificial mixture of BSA, lysozyme, and RNase A. Unexpectedly, the support prepared in the absence of the dye exhibited a separation performance similar to that of the dyed support, contrary to that observed in all previous studies using cyanine dyes as ligands for affinity chromatography, which prompted us to try to determine the structural molecular constitution of the matrix surface. A synthetic route to the final chromatographic support could be devised, which is believed to consist in the cyclization of two nearby ethylenediamine units, involving the inclusion of a succinimide-derived residue between them and the EDC-mediated Lossen rearrangement of an intermediary hydroxamic acid.

     

Affinity chromatography of B6-vitamin-dependent enzymes: purification of pig-heart branched-chain amino acid transaminase

Pig-heart branched-chain amino acid transaminase (EC 2.6.1.42) was purified to near homogeneity with a yield of 27%. A prepurification was performed by heat treatment, gel chromatography and DEAE-Sepharose methods. For the final step, several affinity gels were tested and the one containing cycloserine coupled to CNBr-activated Sepharose 4B was selected. This effected an additional five-fold purification with a yield of 60%. The present affinity results are compared with corresponding studies with other aminotransferases in an attempt to find possible universal techniques for their purification.     

High-performance catalytic chromatography. The adapter approach

A sequence-specific DNA that binds EcoRI endonuclease was immobilized on glycidioloxypropyl-silica and Sepharose by cyanogen bromide (CNBr)-activated coupling. Elution of bound enzyme by conventional affinity strategies (increase of salt concentration) or by catalysis-induced elution (adding a Mg2+ cofactor required for catalysis) was compared. Greater yield and fold-purification was obtained with catalysis-induced elution for both DNA-silica and DNA-Sepharose columns, and silica gives higher performance than Sepharose. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed primarily a single band for EcoRI endonuclease for catalysis-induced elution from DNA-silica columns. Since catalysis-induced elution decreases the lifetime of DNA affinity columns, an alternative approach for preparing re-usable DNA columns was also developed. In this approach, a single stranded adapter DNA sequence is first coupled to silica or Sepharose and then annealed with another DNA sequence that contains a complementary, single stranded tail and the duplex binding site for EcoRI endonuclease. After use, replacing the hydrolyzed DNA regenerates the column. For this adapter approach, Sepharose gives better purity than silica and comparable yields and catalytic based elution gave the highest purity and yield, regardless of support. Substrate DNA with either a tail (for annealing to the column) at one end or both ends were compared and the former gave higher purity. Finally, enzyme binding to the substrate in solution ("trapping") or on a pre-bound substrate column was compared and trapping gave higher yield and similar purity to the alternative. Thus, trapping with a single tailed substrate oligonucleotide on a Sepharose adapter column and using catalytic elution gave the highest performance.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low‐cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE‐4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low‐cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.     

Purification of soluble acetylcholinesterase from sheep liver by affinity chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium’s pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A–Sepharose 4B and edrophonium–Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21 U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.     

Favourable biospecific reactivity of blood group B antigenic trisaccharide chemically attached to poly-N-(2-hydroxyethyl)acrylamide-coated porous glass.

Blood group B antigenic trisaccharide-beta-aminopropyl glycoside (B-TSAP) covalently attached to poly-N-(2-hydroxyethyl)acrylamide-coated porous glass interacts with anti-B monoclonal antibodies faster than the ligand coupled to CNBr-activated Sepharose 4B and Affi-Gel 10. Rates of hydrophobic adsorption of antibodies on the butyl derivatives of the same supports were measured to evaluate the diffusion input to overall kinetics. The lowest average affinity adsorption time [t1(aff) = 250 s] observed for polymer-coated glass probably arises because of the flexibility of the extended segments of chemisorbed N-substituted polyacrylamide acting as effective spacer arms.     

分离尿激酶的亲和色谱填料的制备

 合成了分别以 Sepharose和聚甲基丙烯酸环氧丙酯为基质、对氨基苯甲脒为配基的分离尿激酶的两种亲和色谱填料 ,并用于尿激酶粗品的直接纯化 ,活性回收率分别为 1 0 8.3 %和 43 .4% ,比活提高倍数分别为 9.0 6倍和 3 6.9倍。     

4-(1H-imidazol-1-yl) aniline: A new ligand of mixed-mode chromatography for antibody purification

4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.     

Affinity chromatography of porcine pepsin on different types of immobilized 3,5-diiodo-L-tyrosine

The preparation of affinity sorbents containing immobilized iodinated derivatives of L-tyrosine for the affinity chromatography of porcine pepsin is described. The ligand was coupled either to Sepharose 4B or bead cellulose after the divinylsulfone activation or to Sepharose 4B after the activation with 2,4,6-trichloro-1,3,5-triazine. The highest capacity for porcine pepsin was found in the case of 3,5-diiodo-L-tyrosine coupled to divinylsulfone-activated Sepharose.