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1.
We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.  相似文献   
2.
The heparin-loaded microcapsules were successfully prepared by layer-by-layer deposition of chitosan (CHI) and heparin (Hep). Film growth was confirmed by the reversal of ζ-potentials during polysaccharide deposition. Both scanning electron microscopy and transmission electron microscope evidenced the integrity of (CHI/Hep)5 capsules after the removal of cores. By assembling the carriers with chitosan that are inherently degradable, the capsules were engineered to degrade specifically in the presence of lysozyme. It was demonstrated that the loaded heparin was released from the capsules over a long period of time when being incubated in lysozyme solution. With these results, such CHI/Hep capsules may have a great potential as controlled release carrier for heparin.  相似文献   
3.
以沉淀法制备了正癸酸修饰磁性纳米Fe3O4,采用XRD、TEM和FT-IR对修饰前后的磁性纳米粒子的形态、结构进行了表征。将修饰后的磁性纳米粒子用于对溶菌酶蛋白进行吸附分离,研究了溶液的pH、温度、时间、溶菌酶初始浓度、离子强度等因素对吸附过程的影响。结果表明:pH=10.7,吸附温度为25℃,吸附时间为2.0 h,溶菌酶初始浓度为0.30 mg·mL-1,最大吸附容量为35.0 mg·g-1。修饰后的磁性纳米粒子用于从鸡蛋清中提取溶菌酶,纯化倍数为30.9,酶活力收得率为73.0%。  相似文献   
4.
As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 μL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.  相似文献   
5.
The core–shell structure Fe3O4/SiO2 magnetic microspheres were prepared by a sol–gel method, and immobiled with iminodiacetic acid (IDA) as metal ion affinity ligands for protein adsorption. The size, morphology, magnetic properties and surface modification of magnetic silica nanospheres were characterized by various modern analytical instruments. It was shown that the magnetic silica nanospheres exhibited superparamagnetism with saturation magnetization values of up to 58.1 emu/g. Three divalent metal ions, Cu2+, Ni2+ and Zn2+, were chelated on the Fe3O4@SiO2–IDA magnetic microspheres to adsorb lysozyme. The results indicated that Ni2+‐chelating magnetic microspheres had the maximum adsorption capacity for lysozyme of 51.0 mg/g, adsorption equilibrium could be achieved within 60 min and the adsorbed protein could be easily eluted. Furthermore, the synthesized Fe3O4@SiO2–IDA–Ni2+ magnetic microspheres were successfully applied for selective enrichment lysozyme from egg white and His‐tag recombinant Homer 1a from the inclusion extraction expressed in Escherichia coli. The result indicated that the magnetic microspheres showed unique characteristics of high selective separation behavior of protein mixture, low nonspecific adsorption, and easy handling. This demonstrates that the magnetic silica microspheres can be used efficiently in protein separation or purification and show great potential in the pretreatment of the biological sample. Copyright © 2015 John Wiley & Sons, Ltd.  相似文献   
6.
The multifarious injection chamber for molecular structure study (MICOSS) experimental system has been developed at the Pohang Accelerator Laboratory X‐ray Free‐Electron Laser for conducting serial femtosecond crystallography. This system comprises several instruments such as a dedicated sample chamber, sample injectors, sample environment diagnostic system and detector stage for convenient distance manipulation. Serial femtosecond crystallography experiments of lysozyme crystals have been conducted successfully. The diffraction peaks have reached to ~1.8 Å resolution at the photon energy of 9.785 keV.  相似文献   
7.
A novel, facile, and robust strategy was proposed to increase the pore size and mechanical strength of cryogels. By mixing the monomers of acrylamide and 2‐hydroxyethyl methacrylate as the precursor, a monolithic copolymer cryogel with large interconnected pores and thick pore walls was prepared. Hydrogen bonding between the two monomers contributed to the entanglement and aggregation of the copolymers, thickening the pore walls and resulting in larger pore sizes. Analysis via mercury porosimetry demonstrated that the interconnected pore diameter of the copolymer cryogel ranged from 10‐350 µm, which was far larger than that of the cryogels from one monomer (10‐50 µm). Additionally, the thicker pore walls of the copolymer cryogel improved its mechanical strength. Affinity cryogels were prepared through covalent immobilization using Tris(hydroxymethyl)aminomethane as a coupling agent, and the affinity binding of lysozymes on Tris‐cryogel was evaluated by the Langmuir isothermal adsorption with the maximum adsorption capacity of 360 mg/g. Compared with that of the Tris‐cryogels produced from one monomer, the copolymer Tris‐cryogel exhibited higher adsorption capacity and lysozyme purity, when the chicken egg white solution flowed solely driven by gravity. This work provides a new avenue for designing and developing supermacroporous cryogels for bioseparation.  相似文献   
8.
High-pressure treatment, e.g. used as an alternative method for food preservation, affects protein cross-linking and glycation reactions. These reactions were monitored by using mainly milk proteins in the absence and presence of different saccharides or dicarbonyl compounds. Without carbohydrates, protein cross-linking of casein is enhanced by pressure through the formation of dehydroalanine-derived lysinoalanine. A similar effect can be observed in wool, where pressure accelerates the formation of lanthionine. In contrast, saccharide or dicarbonyl compound-induced cross-linking is constrained by high pressures. Despite this, pressure is able to accelerate the degradation of sugar or carbonyl compounds, as could be seen by the measurement of their residual contents in the examined test preparations.  相似文献   
9.
Protein structure and dynamics can be characterized on the atomistic level with both nuclear magnetic resonance (NMR) experiments and molecular dynamics (MD) simulations. Here, we quantify the ability of the recently presented CHARMM36 (C36) force field (FF) to reproduce various NMR observables using MD simulations. The studied NMR properties include backbone scalar couplings across hydrogen bonds, residual dipolar couplings (RDCs) and relaxation order parameter, as well as scalar couplings, RDCs, and order parameters for side‐chain amino‐ and methyl‐containing groups. It is shown that the C36 FF leads to better correlation with experimental data compared to the CHARMM22/CMAP FF and suggest using C36 in protein simulations. Although both CHARMM FFs contains the same nonbond parameters, our results show how the changes in the internal parameters associated with the peptide backbone via CMAP and the χ1 and χ2 dihedral parameters leads to improved treatment of the analyzed nonbond interactions. This highlights the importance of proper treatment of the internal covalent components in modeling nonbond interactions with molecular mechanics FFs. © 2013 Wiley Periodicals, Inc.  相似文献   
10.
Abstract

The lysozyme- and DNA-binding affinities of the biologically active cisplatin analogues, Pd(II) (1) and Pt(II) (2) complexes of functionalized N,N-pyridylbenzimidazole ligand, are reported. The electronic transitions were studied by time-dependent density functional theory. Complex 1 exhibits interesting antifungal activity against Candida albicans (MIC?=?32?μg?mL?1; 64?nM) and Cryptococcus neoformans (MIC?=?16?μg?mL?1, 32?nM). Complex 1 is covalently bound to DNA and lysozyme via the elimination of the chloride ligand(s). When complex 2 interacts with lysozyme, two adduct peaks are observed in the mass spectrum corresponding to binding of Pt(II) ion to lysozyme.  相似文献   
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