Screening of peptide affinity tags using immobilised metal affinity chromatography in 96-well plate format

A method for high throughput screening of Green Fluorescent Proteins carrying metal binding tags in bacteria was developed. A random four amino acids tag-peptide library was successfully generated in E. coli. A 96-microtiter plate assembled with metal-iminodiacetic acid small cryogel columns was used for library screening. For the first time we were able to simultaneously screen a metal binding peptide tags library obtained from E. coli against different metal ions. From screening 25 different tags, three clones were able to bind to all metal ions studied (Ni2+, Zn2+, Co2+ and Cd2+). It was clearly demonstrated that the new construct could facilitate the screening of large peptide libraries.     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

Specific capture of uranyl protein targets by metal affinity chromatography

To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.     

Searching for cyclin-dependent kinase inhibitors using a new variant of the cope elimination

beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope-type elimination to a vinylsulfone that can be captured by amines to afford beta-aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4-position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2. One of the most potent inhibitors (IC50 = 45 nM) contained a N-3-hydroxypropyl group on the aminoethylsulfonyl substituent. The crystal structure of this inhibitor bound to CDK2/cyclin A was determined and shows an unusual network of hydrogen bonds. The synthetic methodology developed can be utilized in multiple-parallel format and has numerous potential applications in medicinal chemistry.     

Fragment screening of cyclin G-associated kinase by weak affinity chromatography

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K (D))?=?mM?-?μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46?%) with K (D)?相似文献   

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).     

Preconcentration of the herbicide glyphosate and its metabolite AMPA by immobilised metal ion affinity chromatography (IMAC)

A method based on Immobilised Metal Ion Affinity Chromatography (IMAC) using a chelating resin (Spheron Oxine 1000) loaded with Pd(II) is evaluated for the extraction and preconcentration of glyphosate and AMPA from natural water samples. The efficiency of the metal-loaded resin in retaining glyphosate and AMPA and the ability of different reagents to recover them is investigated. The most effective elution solution for the recovery of both analytes from the resin is found to be a mixture of 0.1 M HCI and 1 M NaCl. The effect of flow rate, analyte concentration, and sample volume is evaluated. The optimised experimental conditions are then used in the extraction of the analytes from spiked natural waters. The use of the Pd loaded resin led to recoveries ranging from 80-90% for glyphosate and 60-80% for AMPA.     

Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries

The affinity inhibitor of fusion peptide of influenza A virus has been studied using a combination of high-performance affinity chromatography (HPAC) and combinatorial peptide libraries. Fusion peptide (FP) (1-11) of influenza A virus was used as the affinity ligand and immobilized onto the poly(glycidyl methacrylate) (PGMA) beads. Positional scanning peptide libraries based on antisense peptide strategy and extended peptide libraries were designed and synthesized. The screening was carried out at acidic pH (5.5) in order to imitate the environment of virus fusion. A hendecapeptide FHRKKGRGKHK was identified to have a strong affinity to the FP (1-11). The dissociation constant of the complex of the hendecapeptide and the FP (1-11) is 3.10 x 10(-6) mol l(-1) in a physiological buffer condition. The polypeptide has a fairly inhibitory effect on three different strains of influenza A virus H1N1 subtype.     

Fishing DNA targets in DNA solutions by using affinity microcontact printing

In this paper, we report the application of affinity microcontact printing (αCP) for "fishing" DNA targets in aqueous solutions and transferring them to solid surfaces for detection purposes. Affinity stamps used in this experiment were made of poly(dimethylsiloxane) (PDMS) with DNA probes covalently immobilized on their surface. When these stamps were immersed in DNA solutions, DNA targets with a perfect-match (PM) sequence to the probes can selectively hybridize to the stamp surfaces and then be transferred to solid surfaces. However, to distinguish PM DNA from single base-pair mismatch (1MM) DNA targets, 10 mM of NaCl must be added to the hybridization buffer. Under the optimized conditions, this αCP can lead to a surface density of PM which is 15 times higher than that of 1MM. The affinity stamp is also able to "fish" PM DNA targets from a mixture of PM/1MM DNA targets and transfer them to solid surfaces. Because DNA probes and targets are separated after printing, we also applied this technique for label-free detection of DNA targets by using liquid crystals.     

亲和色谱法筛选中药中血管紧张素转化酶抑制剂

以壳聚糖微球为载体、戊二醛（glutaraldehyde,GA）为交联剂对血管紧张素转化酶（Angiotensin converting enzyme,ACE）进行固定化．用固化的ACE作为亲和介质,利用血管紧张素转化酶抑制剂（Angiotensin convertingenzym einhibitor,ACEI）与ACE之间的亲和作用,结合高效液相色谱对亲和前后的体系进行检测,比较两者各组分色谱峰的差异,以此实现快速筛选复杂体系中的ACE抑制剂．应用赖诺普利（Lisinopril）、九肽抑制剂、依那普利（Enalapril）、培哚普利（Perindopril）、卡托普利（Captopril）等已上市的ACEI对方法进行验证,反映方法具有高度选择性．将方法应用于中药地龙及山楂筛选,发现共有5个组分与ACE有亲和作用,并且都能抑制ACE酶活性,它们对酶活性抑制的IC50值在0．45～4．62μg／mL范围．通过对亲和方法重现性考察,6次测定的相对标准偏差小于1％,说明方法可靠．提出的亲和色谱．色谱指纹差异法非常适合于从中药及天然产物等复杂混合物库中快速筛选靶点活性物质．     

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.     

Fractionation of glycoprotein-derived oligosaccharides by affinity chromatography using immobilized lectin columns

Lectin affinity column chromatography is becoming a method of choice for the fractionation and purification of oligosaccharides, especially N-linked oligosaccharides. Using lectin affinity, it is easy to separate structural isomers and to isolate oligosaccharides based on specific features. Further, serial lectin column chromatography, when various lectin columns are used at the same time, can afford a very sensitive method for the fractionation and characterization of extremely small amounts of oligosaccharides. Thus, when used in conjunction with other separation techniques, lectin affinity chromatography can help to purify rapidly oligosaccharides and provide substantial information about their structural features.     

Virtual target screening: validation using kinase inhibitors

Computational methods involving virtual screening could potentially be employed to discover new biomolecular targets for an individual molecule of interest (MOI). However, existing scoring functions may not accurately differentiate proteins to which the MOI binds from a larger set of macromolecules in a protein structural database. An MOI will most likely have varying degrees of predicted binding affinities to many protein targets. However, correctly interpreting a docking score as a hit for the MOI docked to any individual protein can be problematic. In our method, which we term "Virtual Target Screening (VTS)", a set of small drug-like molecules are docked against each structure in the protein library to produce benchmark statistics. This calibration provides a reference for each protein so that hits can be identified for an MOI. VTS can then be used as tool for: drug repositioning (repurposing), specificity and toxicity testing, identifying potential metabolites, probing protein structures for allosteric sites, and testing focused libraries (collection of MOIs with similar chemotypes) for selectivity. To validate our VTS method, twenty kinase inhibitors were docked to a collection of calibrated protein structures. Here, we report our results where VTS predicted protein kinases as hits in preference to other proteins in our database. Concurrently, a graphical interface for VTS was developed.     

糖原合成酶激酶-3和细胞周期蛋白依赖性激酶抑制剂paullones的研究进展

对抑制剂paullones的来源、合成,以及与激酶相互作用的分子机理、选择性、细胞效应进行了综述.指出糖原合成酶激酶-3(GSK-3)是参与糖代谢的主要限速酶之一,人类多种疾病均与其活性调节异常有关;细胞周期蛋白依赖性激酶(CDKs)是一类丝氨酸/苏氨酸蛋白激酶,能够促使细胞进行有序的生长、增殖、休眠或进入凋亡.以CDKs为靶点的药物可以阻断细胞周期,控制癌细胞增殖,从而达到抗肿瘤的目的.Paullones对CDKs和GSK-3均具有良好的选择性,是CDKs和GSK-3的有效抑制剂;迄今为止,文献报道的paullones化合物接近120个.     

Inverse in silico screening for identification of kinase inhibitor targets

Protein kinases are clinically relevant, attractive drug targets for cancer. One major problem with kinase inhibitors is broad promiscuity, causing off-target actions and side effects. In silico prediction of targets of a compound would immensely facilitate and accelerate drug development. Using a virtual "inverse" screening approach, where single compounds are docked into protein structures from a database, we identify among known targets of indirubin derivatives phosphoinositide-dependent kinase 1 (PDK1) as a target of one derivative (6BIO) in particular. This prediction is functionally supported by an in vitro kinase assay, inhibition of intracellular phosphorylation of PDK1-substrates, and inhibition of endothelial cell migration, which highly depends on PDK1. Virtual inverse screening combined with biological tests, thus, is proposed as a valuable tool for the drug discovery process and re-examination of already established kinase inhibitors.     

Separation of phosphomolybdate by affinity chromatography

A new method of separating phosphomolybdate from phosphoric esters and anhydrides using a small column of polyvinylpolypyrrolidone is introduced. When a mixture of phosphate compounds and ammonium molybdate (1–3%, pH 3-5) is poured onto such a column, inorganic orthophosphate (Pi)^p1 is selectively adsorbed onto the column material as phosphomolybdate, while other phosphate compounds, which do not react with molybdate, are drained through the column. Using radioisotopes, retention and recovery percentages were measured. At pH 3, 99.99% of Pi^p32 was retained in the column, while 97 ± 1% of ATP^p32 was recovered in the effluent. Retained Pi^p32 was eluted out later with 0.5 M ammonium hydroxide with a recovery percentage of 98 ± 1%. Unlike other methods of separating phosphomolybdate, the separation was little affected by the presence of reducing agents. The use of disposable columns, which can be prepared easily, packed ahead of time, and stored for later use, makes the radioisotopes assay convenient and contamination-free.     

Screening for galectin-3 inhibitors from synthetic lacto-N-biose libraries using microscale affinity chromatography coupled to mass spectrometry

The synthesis and screening of two beta-D-Galp-(1-3)-beta-d-GlcpN (lacto-N-biose) disaccharide libraries are reported. Solution-phase synthetic modifications at the HO-2' and NH positions were performed in an effort to enhance the affinity toward galectin-3, a galactose-binding protein involved in tumor metastasis, apoptosis, and inflammation. The libraries were screened for galectin-3 binding by microscale frontal affinity chromatography coupled to mass spectrometry (FAC/MS) allowing for rapid ranking of the different inhibitors and the determination of the galectin-3 binding Kd's. Compounds bearing a hydrophobic substituent on the NH group showed the highest affinity for the lectin. The N-naphthoyl derivative (Kd = 10.6 microM) was the best inhibitor with a 7 times increased affinity as compared to the N-acetyl parent compound (Kd = 73.3 microM).