新多肽配体GE11与表皮生长因子受体的结合能力分析

应用亲和毛细管电泳（ACE）分析方法,对表皮生长因子受体（EGFR）和新多肽配体GE11之间的结合能力进行分析。结果表明,EGFR与多肽配体GE11之间存在特异性相互作用,考察EGFR在不同浓度GE11溶液中的迁移情况,采用非线性、双倒数、Y-倒数和X-倒数4个数据处理方法得到较好的数据拟合,并测得结合常数。该文为筛选多肽配体以及测定受体与多肽配体之间的结合常数提供了简便的方法,将有力推动肿瘤靶向药物输送的研究。     

Screening and chromatographic assessing of a novel IgG biomimetic ligand

A novel biomimetic ligand, N-benzyloxycarbonyl-l-tyrosine (N-cbz-l-Tyr), was screened by a combination method of molecular docking and immobilized receptor technique. Then, N-cbz-l-Tyr was immobilized on Sepharose CL-4B to prepare a specific affinity adsorbent for immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on the adsorbent gave an association constant (K(a)) of 4.91 x 10(6) m(-1) and a theoretical maximum adsorption capacity of 17.3 mg IgG/mL gel. IgG with a purity of 98% was separated from human plasma by this new affinity adsorbent.     

Identification of impurities in acarbose by using an integrated liquid chromatography-nuclear magnetic resonance and liquid chromatography-mass spectrometry approach

The usefulness of applying an integrated LC-NMR and LC-MS approach to acarbose bulk drug impurity profiling is demonstrated. LC-MS and LC-NMR methodologies were employed for the online separation and structural elucidation of a final drug product. Combining data provided by the stop-flow LC-NMR and LC-MS experiments made it possible to identify the main components present in the acarbose sample. Spectral analysis revealed that A and B were known impurities while C was an unknown compound. LC-MS and LC-NMR analyses revealed that C was a pentasaccharide differing from the acarbose in number and nature of sugar subunits in the molecule. It was subsequently isolated and its structure was confirmed by the offline 1- and 2-D NMR experiments, and atom assignment was made.     

Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation

Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.     

Development and screening of epoxy‐spacer‐phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide‐expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost‐effective one‐step chromatographic set‐up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di‐amino‐alkanes (C₂–C₁₀), and branched polyethyleneimine spacers with different molecular weights (800 Da–10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15‐mer and a cyclic 6‐mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.     

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ～17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.     

Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip

A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.     

Flexible docking simulations: Scaled collective variable Monte Carlo minimization approach using Bezier splines,and comparison with a standard Monte Carlo algorithm

An algorithm for docking a flexible ligand onto a flexible or rigid receptor, using the scaled‐collective‐variables Monte Carlo with energy minimization approach, is presented. Energy minimization is shown to be one of the best techniques for distinguishing between native‐ and nonnative‐generated conformations. Incorporation of this technique into a Monte Carlo procedure enables one to distinguish the native conformation directly during the conformational search. It avoids the generation of a large number of ligand conformers for which more sophisticated energy evaluation tools would have had to be applied to identify the nativelike conformations. The efficiency of the Monte Carlo minimization was greatly improved by incorporating a new grid‐based energy evaluation technique using Bezier splines for which the energy function, as well as all of its derivatives, can be deduced from the values at the grid points. Comparison between our ECEPP/3‐based algorithm and the Monte Carlo algorithm presented elsewhere (Hart, T. N.; Read, R. J. Prot Struct Funct Genet 1992, 13, 206–222) has been made for docking NH₂ D Phe Pro Arg COOH, the noncovalent analog of NH₂ D Phe Pro Arg chloromethylketone (PPACK), onto the active site of human α‐thrombin. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 244–252, 1999     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A,a selective kappa opioid receptor agonist

Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based “agonist-bound” hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental pK _i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

高效亲和色谱研究含有特定结构域肽与DNA的结合

高效亲和色谱研究含有特定结构域肽与ＤＮＡ的结合张若蘅,杨栩,李崇熙,徐筱杰（北京大学化学系北京１００８７１）１前言在脱氧核糖核酸（ＤＮＡ）结合蛋白质中,除了螺旋－转折。螺旋、锌指和亮氨酸拉链三种主要结构域之外￣［１］,近年来,一类含有β－转折结构的Ｓ...     

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.     

Efficient estimation of binding free energies between peptides and an MHC class II molecule using coarse‐grained molecular dynamics simulations with a weighted histogram analysis method

We estimate the binding free energy between peptides and an MHC class II molecule using molecular dynamics (MD) simulations with the weighted histogram analysis method (WHAM). We show that, owing to its more thorough sampling in the available computational time, the binding free energy obtained by pulling the whole peptide using a coarse‐grained (CG) force field (MARTINI) is less prone to significant error induced by inadequate‐sampling than using an atomistic force field (AMBER). We further demonstrate that using CG MD to pull 3–4 residue peptide segments while leaving the remaining peptide segments in the binding groove and adding up the binding free energies of all peptide segments gives robust binding free energy estimations, which are in good agreement with the experimentally measured binding affinities for the peptide sequences studied. Our approach thus provides a promising and computationally efficient way to rapidly and reliably estimate the binding free energy between an arbitrary peptide and an MHC class II molecule. © 2017 Wiley Periodicals, Inc.     

Molecular Modeling of Interactions of the Benzolactam-V8 Modulators with the Cys2 Domain of Protein Kinase Cδ

Molecular modeling of interactions of four 7- or 8-substituted benzolactam-V8 （BLV） molecules with the cys2 activator-binding domain of protein kinase C （PKCδ） was carried out using molecular docking program Autodock. The docked models reveal that the hydroxymethyl group at the C（5） atom of the eight-membered ring of each BLV is bound at the bottom of the binding groove of the cys2 domain of PKCδ The BLV molecules make hydrogen bonds and hydrophobic interactions with PKCδ, which are similar to those in the crystal structure of the cys2 domain of PKCδ in complex with phorbol 13-acetate. BLV-1 does not contain a long side chain that is hydrophobic and necessary for membrane insertion, so that it would not be a potent modulator of PKCδ. The other three BLV molecules have long side chains substituted at C（7） or C（8） atoms, and it was predicted, based on the docking results, that they had the PKCδ-binding affinity in the order of BLV-2〉BLV-4〉BLV-3, and BLV-2 would be a potent activator of PKCδ.     

Binding constant determination of high-affinity protein-ligand complexes by electrospray ionization mass spectrometry and ligand competition

We describe an approach for the determination of binding constants for protein-ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen-activated protein kinase.     

Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.     

MeCAT peptide labeling for the absolute quantification of proteins by 2D‐LC‐ICP‐MS

Metal‐Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP‐MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two‐dimensional separations based on strong cation exchange (SCX) and reversed‐phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion. The elution protocols were optimized to improve the resolution of the MeCAT‐labeled peptides which led to faster elutions in SCX and longer retention times in RP compared with unlabeled peptides. The optimized method provided enough resolution for the samples analyzed. Peptides losses during the whole procedure were studied. Although recoveries of greater than 90% were found in the RP dimension, important global losses in the two‐dimensional offline approach forced us to use specific internal standards, in this case MeCAT‐labeled standard peptides. External calibration and label‐specific isotope dilution analysis (IDA) were tested and compared as possible quantification techniques. While both techniques showed accurate and precise determinations, the label‐specific IDA technique resulted in more straightforward measurements and more affordable external calibrations. Finally, simultaneous quantification of three different samples labeled with different lanthanides was successfully performed demonstrating the potential of MeCAT combined with ICP‐MS for multiplexing. Electrospray ionization mass spectrometry techniques provided the structural information needed for the identification of the labeled species. Copyright © 2012 John Wiley & Sons, Ltd.     

Preliminary study on application of impregnated synthetic peptide TLC stationary phases for the pre-screening of 5-HT1A ligands

Chromatographic parameters (deltaR(f)), defined as a difference in the migration of tested compound on the control and peptide impregnated silica gel TLC plates, were determined for 42 arylpiperazine derivatives. An amino acid sequence of the peptide used for impregnation was derived from the III transmembrane segment of the 5-HT(1A) receptor in the close vicinity of aspartic acid (Asp 166) residue. It was found that the deltaR(f) values obtained in a model employing tetrapeptide P4LA (ADVL), as well as the calculated logP correlate with 5-HT(1A) receptor affinity of the studied compounds.