Separation of non-denatured proteins using semi-crosslinked polyacrylamide capillary gel electrophoresis

This work presents an approach to build a high-performance, low-viscous and replaceable separation matrix, semi-crosslinked polyacrylamide （semi-CPA） capillary gel electrophoresis. Non- denatured basic proteins, such as lysozyme, cytochrome C, ribonuclease A and trypsin were separa- ted. The impacts of monomer and cross-linker concentrations on protein separation were studied, and the ability of dynamic capillary inner wall coating was demonstrated. The UV absorption interfer- ence by semi-CPA gel matrix was successfully overcome by a partial filling technique, which results in sensitivity 20 times higher than other protein separation method. The excellent separation ability, reproducibility and dynamic coating ability made semi-CPA an ideal separation media in both capillar- y electrophoresis and microfluidic chip separation scheme.

Fe_3O_4@PAM@NTA-Ni^(2+) Magnetic Composite Nanoparticles for Highly Specific Separation of His-tagged Proteins

A facile approach has been developed to synthesize Fe3O4@PAM（polyacrylamide） nanoparticles（NPs） with carboxyl groups on the surfaces by copolymerization with acrylamide and acrylic acid in Fe3O4 NPs aqueous suspension. Nitrilotriacetic acid（NTA） was conjugated to the magnetic NPs via well-known carboniimide chemistry using EDC and NHS. The Ni^（2＋） ions loaded on the surface of NPs provide abundant docking sites for immobilization of His-tagged green fluorescent proteins（His-tagged GFP）. The high magnetic property of Fe3O4@PAM@NTA-Ni^（2＋） allows an easy separation of the NPs from solution under an external magnetic field, with high His-tagged protein binding capacity（42 μg protein/mg of NPs）. The NPs can be recycled for at least four times without significant loss of binding capacity to proteins. These materials show great potential to separate His-tagged protein with low-cost purification at industrial scale.

High Performance Hydrophobic Interaction Chromatography-A New Approach to Separate Intermediates of Protein Folding──Ⅰ.Separation of Intermediates of Urea-unfolded α-Amylase

Based on the different hydrophobicities of the intermediates of proteins the various conformational intermediates of the refolding of a-amylase originally denatured with 8.0 mol/L urea solution were separated with high performance hydrophobic interaction chromatography(HPHIC). Compared to the separation of the same intermediates with weak anion exchange chromatography and size-exclusion chromatography the result obtained with HPHIC is the best It would be expected that HPHIC may be a strongly potential tool to separate intermediates of some proteins which cannot be, or cannot completely be refolded by HPHIC.

New Development of Reverse Micelles and Applications in Protein Separation and Refolding

反向的微粒在器官的溶剂带温和、有效的微型环境包含简历分子，它在蛋白质，蛋白质 refolding，和酶的反应的隔离为申请吸引了巨大的注意。在这评论，为蛋白质分离和 refolding 的反向的微粒的应用程序简短被总结了，各种各样的反向的 micellar 系统不同表面活化剂创作了，包括离子， nonionic，混合，并且基于亲密关系的反向的微粒，被加亮。它特别说明由结合的 biocompatible 表面活化剂组成的新奇基于亲密关系的反向的微粒的潜在的申请亲密关系 ligands。而且，在生物工学的下游的处理为蛋白质分离和 refolding 开发通用基于亲密关系的反向的微粒的重要性被指出了。

The pathogenic mechanism of TAR DNA-binding protein 43(TDP-43)in amyotrophic lateral sclerosis

The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).

Capillary Electrophoretic Separation of Proteins under the Control of Radial Electric Field

Separation of basic proteins was performed using a homemade field-modulated capillary electrophoresis system. The resolution. elution and even wall adsorption can be regulated by ad-lusting the radial rather than axial voltage applied. Selection of running buffer and pH was found to be critical.

Capillary Zone Electrophoretic Separation of Basic Proteins with Coated Columns Prepared by Sol-Gel Technology

Coated capillary columns were prepared by sol-gel technology and used in the separation of basic proteins with capillary zone electrophoresis. The results indicated that a significant decrease in protein adsorption was obtained and EOF was also diminished to zero in the pH range of 3-10.

Selective separation and enrichment of proteins in aqueous two-phase extraction system

木钉 / 磷酸盐的一个简单水的二阶段的抽取系统(ATPS ) 为蛋白质的选择分离和丰富被建议。有 HPLC 的 ATPE 的联合被使用在二个阶段识别蛋白质的分区。五蛋白质(牛的浆液白朊，细胞色素 C，溶解酵素，肌球素，和胰岛素) 被用作模型蛋白质在蛋白质分区上学习磷酸盐集中和 pH 的效果。木钉 / 磷酸盐系统第一被用于真实人的口水和血浆样品，一些蛋白质在二个阶段显示出显然不同的分区。主要结果在提供的 ATPS 表明蛋白质的选择分离和丰富为在 proteomics 的高丰富蛋白质弄空的潜力。

SSINCC: Simple separation of interacting nucleoprotein complex components

Protein-DNA binding assays have been used in a va-riety of applications from fundamental studies re-garding the binding process itself to serve as probes for the detection, quantification and separation of target analytes. Here we describe a novel method of analyzing and identifying intermolecular DNA interactions that allows for the simple separation of interacting nucleoprotein complex components (SSINCC), focusing specifically on DNA-DNA interactions using P1 plasmid active partition system nucleoprotein complexes as a model to demonstrate DNA sequence specificity and tolerance of composite factor complexity. Traditional and recent assays of protein-DNA interaction are summarized and compared with SSINC. Although SSINC is examined here employing P1 partition nucleoprotein complex as an example of DNA-DNA intermolecular association, universal applications of this methodology to nucleo-protein complex studies can be envisioned.

Plant RNA-binding proteins:Phase separation dynamics and functional mechanisms underlying plant development and stress responses

RNA-binding proteins(RBPs)accompany RNA from synthesis to decay,mediating every aspect of RNA metabolism and impacting diverse cellular and developmental processes in eukaryotes.Many RBPs undergo phase separation along with their bound RNA to form and function in dynamic membraneless biomolecular condensates for spatiotemporal coordination or regulation of RNA metabolism.Increasing evidence suggests that phase-separating RBPs with RNA-binding domains and intrinsically disordered regions play important roles in plant development and stress adaptation.Here,we summarize the current knowledge about how dynamic partitioning of RBPs into condensates controls plant development and enables sensing of experimental changes to confer growth plasticity under stress conditions,with a focus on the dynamics and functional mechanisms of RBP-rich nuclear condensates and cytoplasmic granules in mediating RNA metabolism.We also discuss roles of multiple factors,such as environmental signals,protein modifications,and N6-methyladenosine RNA methylation,in modulating the phase separation behaviors of RBPs,and highlight the prospects and challenges for future research on phase-separating RBPs in crops.

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 μmol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE mass spectrometry (CE MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE MS.

Separation of Recombinant Geranylgeranyl Diphosphate Synthase of Deinococcus radiodurans from Expressed Strain Cell Homogenate by Immobilized Metal Affinity Chromatography on a Characterized Monolithic Cryogel Column

Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the biosynthesis of antioxidative ca-rotenoid from the extremely radioresistant bacterium Deinococcus radiodurans. In this work, the recombinant GGPPS expressed in Escherichia coli by cloning and transforming the gene dr1395 of D. radiodurans was isolated rapidly by an immobilized metal affinity supermacroporous cryogel, i.e., Cu2+-iminodiacetic acid (IDA)-cryogel. The properties of the Cu2+-IDA-cryogel were characterized using capillary-based mathematical model and experi-mental measurements. The obtained protein samples were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The porosity of the present Cu2+-IDA-cryogel is 90.4% and the water permeabil-ity is 5.04×10-12 m2. From the capillary-based model, this cryogel presents a slightly wide normal pore (capillary) size distribution with the mean diameter of 55.2 μm, the standard deviation of 28.0 μm and the half of skeleton wall thickness of 2.8 μm. The pore size distribute from about 10 to 141 μm and the effective tortuosity of these capillary pores increases from 2.60 to 9.05. The isolation of the GGPPS from cell homogenate can be achieved at the flow velocity of 3.40×10-4 m·s-1 by the Cu2+-IDA-cryogel bed. High-purity GGPPS (about 91.4%) is obtained according to the SDS-PAGE analysis of the elution samples, indicating that the present method is a promising, simple and ef-fective approach to isolate GGPPS from cell homogenate of engineering strains.

The effects of maltodextrin/starch in soy protein isolate–wheat gluten on the thermal stability of high-moisture extrudates

This study aimed to investigate the interaction between maltodextrin/starch of different molecular weight distributions and soy protein isolate (SPI)–wheat gluten (WG) matrix during high-moisture extrusion.Two maltodextrins (dextrose equivalent (DE):10 and 20) and wheat starch were extruded with SPI–WG blend in a system of 65,70,and 75%moisture to investigate their effects on texture and thermal stability.Incorporating 5%maltodextrin (DE10) in the SPI–WG matrix improved the fiber structure and thermal stability.When wheat starch was thoroughly gelatinized during subsequent sterilization,the fiber structure and thermal stability were also improved.It was found that the plasticization caused by small-molecular weight saccharides and enhanced phase separation caused by large-molecular weight saccharides changed the melting temperature of blends and significantly improved the texture and thermal stability of extrudates.

Phase separation of GRP7 facilitated by FERONIAmediated phosphorylation inhibits mRNA translation to modulate plant temperature resilience

Changes in ambient temperature profoundly affect plant growth and performance.Therefore,the molecu-larbasis of plant acclimation to temperature fluctuation is of great interest.In this study,we discovered that GLYCINE-RICH RNA-BINDING PROTEIN 7(GRP7)contributes to cold and heat tolerance in Arabidopsis thaliana.We found that exposure to a warm temperature rapidly induces GRP7 condensates in planta,which can be reversed by transfer to a lower temperature.Cell biology and biochemical assays revealed that GRP7 undergoes liquid-liquid phase separation(LLPS)in vivo and in vitro.LLPS of GRP7 in the cyto-plasm contributes to the formation of stress granules that recruit RNA,along with the translation machinery component eukaryotic initiation factor 4E1(elF4E1)and the mRNA chaperones COLD SHOCK PROTEIN 1(CSP1)and CSP3,to inhibit translation.Moreover,natural variations in GRP7 affecting the residue phos-phorylated by the receptorkinase FERONIA alter its capacity to undergo LLPS and correlate with the adap-tation of some Arabidopsis accessions to a widertemperature range.Taken together,ourfindings illustrate the role of translational control mediated by GRP7 LLPS to confer plants with temperature resilience.

“Grafting from”法制备高载量大孔弱阳离子交换层析介质

针对大孔聚合物层析介质孔径大、比表面积低而导致的蛋白结合容量低的问题,采用“graftingfrom”策略,以大孔聚丙烯酸酯微球为基质,通过氧化还原引发甲基丙烯酸在微球表面接枝聚合,制备了高载量弱阳离子交换层析介质.研究了单体浓度、过硫酸钾浓度及反应温度等因素对蛋白结合容量的影响.所得介质的蛋白静态结合容量和动态结合容量分别达到252.21和157.25mg/mL;同时发现具有一定离子交换容量的该类介质能够在0.2 mol/L NaCl缓冲液中保持100 mg/mL的蛋白结合容量.将该层析介质用于鸡卵清中溶菌酶的纯化,获得了较高的纯化效率.

榛仁水解蛋白肽分离纯化及其体外功能活性筛选

采用超滤膜分离、葡聚糖凝胶G-10分离和反相高效液相层析3种逐级递进的分离方法对榛仁水解蛋白肽进行分离纯化,然后以DPPH自由基清除率、羟基自由基清除率和牛血清白蛋白变性抑制率为指标,对分离纯化后的各级组分进行体外活性筛选,得到抗氧化活性最优组分。结果表明:经葡聚糖凝胶G-10分离得到的Z-Z1-P2组分的抗氧化活性高于其他组分,经反相高效液相层析得到的Z-Z1-P2-R2组分抗氧化活性最强,对DPPH自由基的清除率为94.08%±1.60%,对羟基自由基的清除率为87.98%±1.54%。

实验教学中醋酸纤维素薄膜电泳的条件优化

醋酸纤维素薄膜电泳分离血清蛋白是生物化学实验课中的一项基础实验技术。目前,诸多医学院校基础医学及生物学专业均开设了此项实验。但是由于实验方法的不同,且实验过程中的影响因素较多,学生获得的实验结果也存在多种问题。经过多年实验教学探索,西安交通大学医学部基础医学院生物化学与分子生物系实验教学团队优化了醋酸纤维素薄膜电泳分离血清蛋白的实验方案,增强了实验教学内容的互动性,确保了实验结果的稳定性,使学生更好地掌握了水平电泳分离蛋白质的理论知识并获得了成功的实验验证。

红豆肽的制备、分离纯化及降糖活性研究

目的:筛选红豆蛋白的酶解工艺并研究红豆多肽的降糖活性及构效关系。方法:采用碱沉酸提法提取红豆粗蛋白,以肽得率、水解度、α-葡萄糖苷酶抑制、Fe^(2+)螯合率、DPPH清除率为指标筛选最佳蛋白酶;酶解得到红豆多肽,醇沉多糖后通过DA201-C大孔吸附树脂进行脱盐纯化后,测定多肽的分子量及氨基酸组成来探讨其构效关系。结果:经4种蛋白酶(中性、碱性、风味、复合蛋白酶)水解后,碱性蛋白酶的肽得率为82.95%,水解度为52.67%,实验浓度范围内最佳α-葡萄糖苷酶抑制为72.14%,比其他3种酶均高;85%乙醇醇沉后的肽糖比、α-葡萄糖苷酶抑制率(73.49%)和α-淀粉酶抑制率(31.31%)均最高,脱盐纯化后肽含量高达97.61%,在2 mg/mL时测定对α-葡萄糖苷酶和α-淀粉酶抑制效果,分别为52.49%和14.63%,表明除去多糖后红豆多肽仍具有独立降血糖活性;经测定,红豆多肽以小分子为主,3 kDa以下占比91.40%,其中小于1000 Da占比达到75.28%。纯化后疏水性氨基酸占比约37.28%,其中甘氨酸、丙氨酸、脯氨酸和谷氨酸含量较高。结论:碱性蛋白酶是制备红豆多肽的最佳蛋白酶,分离纯化后的红豆多肽具有降糖活性,其活性可能与4种疏水性氨基酸含量较高且分子量较小有关。

眼镜蛇毒细胞毒素-1的分离纯化及其对HSC-LX2细胞PI3K/AKT 信号通路的影响

肝纤维化是多种刺激诱导的肝脏反复损伤的一种病理再生反应[1],其主要表征是肝脏中沉积着过量的细胞外基质(extracellular matrix,ECM)。ECM产生主要原因之一是由于肝星状细胞(hepatic stellate cell,HSC)被大量激活分化,磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)信号通路是一条重要的细胞内信号通路,能通过影响HSC的增殖和凋亡来调节肝纤维化[2-4]。

阳离子化无定形碳酸钙加填纸的制备及其蛋白分离功能

本研究采用液相沉积法,以氯化钙和碳酸钠为原料,引入氯化镁作为晶型稳定剂,成功合成了无定形碳酸钙(amorphous calcium carbonate,ACC)。对ACC进行阳离子化改性,以增强其正电性和分散性。结果表明,阳离子化ACC具有良好的吸附性能;当阳离子试剂质量浓度为2.0 mg/mL,制备的阳离子化无定形碳酸钙加填量为40%时,对牛血清白蛋白的吸附量为92 mg/g。