多聚组氨酸融合标签在蛋白药物开发中的应用

纯化技术是制约蛋白质药物开发及其产业化的关键技术之一。构建多聚组氨酸标签融合蛋白,采用固定金属离子亲和层析进行纯化,是一种高效的蛋白质纯化策略。介绍多聚组氨酸融合标签在蛋白质药物开发中的应用基础和应用概况,分析多聚组氨酸标签在融合蛋白中的位置对亲和层析纯化的影响,总结常用的多聚组氨酸融合表达方式,并对其融合表达样品的预处理、亲和层析纯化条件及其对目的蛋白药物药用安全性和有效性的影响进行探讨。     

基于质谱技术的蛋白质互作研究进展

蛋白质作为生命活动的执行者,其功能往往体现在与其他蛋白质的相互作用中,研究蛋白-蛋白相互作用对于人们深入了解和预防传染病、靶向治疗多基因疾病、阐明蛋白质的分子作用机制及各种复杂的生命现象具有重要意义。目前,有多种技术被用来研究蛋白间的相互作用,研究难点在于实时捕获瞬时或弱蛋白质间的相互作用,质谱技术（mass spectrometry, MS）可在某种程度上解决该难点。由于质谱技术可研究简单的蛋白质复合物再到大规模的蛋白质组实验,基于质谱技术研究蛋白质间相互作用被越来越多地应用于科学研究中。综述了蛋白质间相互作用检测方法的研究进展,重点介绍了氢氘交换质谱法和化学交联质谱法研究蛋白质间相互作用的优缺点及其应用,最后对基于质谱技术研究蛋白质间相互作用进行了总结与展望,以期为深入开展相关研究提供借鉴。     

衰老相关的蛋白稳态失衡

健康细胞利用一系列蛋白质质量调控网络来维持自身蛋白质组的稳定性和功能性,即维持蛋白稳态。但是在衰老过程中普遍出现蛋白稳态失衡的现象,其主要表现是蛋白质合成、折叠和降解之间的平衡被破坏。造成衰老相关蛋白稳态失衡的原因主要有：(1)应激反应相关途径的转录受到抑制;(2)蛋白酶体活性降低和自噬功能出现障碍;(3)核糖体翻译暂停。另外,在衰老过程中细胞主要通过蛋白稳态网络的分子伴侣、蛋白酶体、自噬系统等对蛋白稳态进行调节。本文对衰老过程中造成蛋白稳态失衡的诱因以及蛋白稳态调控的途径进行综述,以期为衰老研究和解决老年健康问题开拓新思路。     

特异性的蛋白小分子荧光探针及其标记技术

活体蛋白荧光标记技术已经被广泛应用于蛋白质功能的可视化研究中。荧光蛋白常被用来研究蛋白质在生物体内的表达和定位,但由于它本身体积比较大,往往会影响目标蛋白的生物活性。特异性的小分子荧光探针以其体积小、膜透性好、背景噪音低以及制备方便的优点成为蛋白质研究的一个有力工具。本文将简要介绍近几年来各类特异性小分子蛋白荧光探针的研究进展。     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

牛乳碱性蛋白二维凝胶电泳条件的优化

为了在二维凝胶电泳(2-DE)中有效分离牛乳中的碱性蛋白,本试验在等电聚焦上样缓冲液中加入异丙醇和甘油,采用杯上样方式,比较了三丁基磷(TBP)和二硫苏糖醇(DTT)两种还原剂对碱性区域蛋白的分离效果。结果发现,等电聚焦上样缓冲液以TBP为还原剂的碱性蛋白分离图谱水平条纹少,优于以DTT为还原剂图谱。表明等电聚焦上样缓冲液以TBP为还原剂,采用杯上样的方法可以改善牛乳中碱性蛋白的分离效果。     

蛋白质内含子介导的蛋白纯化系统在变性条件下的应用

由蛋白质内含子介导的亲和蛋白质纯化系统(IMPACT)已得到广泛应用,通过其纯化得到的目的蛋白不含蛋白纯化标签以及多余的氨基酸残基,且操作简单成本低廉。这些优点使得其相对于其他蛋白纯化系统有着无与伦比的优势。但是现有报道都局限于非变性条件下使用,这往往会限制其在一些包涵体蛋白变性条件下使用。以一已知表达形成包涵体形式的丝素蛋白为例,研究IMPACT系统在时变性条件下使用变性剂浓度、温度和诱导断裂还原剂浓度。实验表明,在4 M尿素,100 mM DTT室温作用下蛋白质内含子会获得最大断裂效率(80%)。柱上在线断裂实验表明,其最终蛋白得率超过65%。     

蛋白质突变库高通量表达筛选方法的研究

人工进化方法被越来越多地应用于重组蛋白的表达筛选及其结构功能的研究,在适当的选择压力下,它可以有效地改善野生型蛋白固有的低表达和低稳定性等问题。本研究就如何进行快速、有效的蛋白质人工进化以提高重组蛋白表达量,进行了方法学探索,通过建立以绿色荧光蛋白为报告基因、结合流式细胞仪分选和96孔板等高通量筛选方法,成功地进行了一例高通量蛋白质点突变库重组表达株的筛选。     

基于物化性质对嗜热蛋白的预测

嗜热蛋白在高温下能保持稳定性和活性,是研究蛋白质热稳定性的理想模型,开发一个蛋白质热稳定性识别的方法将对蛋白质工程和蛋白质的设计很有帮助。目前的研究中,氨基酸的组成及其物化性质一直被认为和蛋白质的热稳定性相关。本研究筛选出可靠的数据集,包括915个嗜热蛋白和793个非嗜热蛋白。利用蛋白质氨基酸的物化性质和氨基酸的组成表征嗜热蛋白,将二肽氨基酸组成整合到9组氨基酸物化性质中使蛋白序列公式化。支持向量机5折叠交叉验证表明:当gap=0时,290个特征产生的精度最高,为92.74%。因此说明对于分析蛋白质的热稳定性,所建立的预测模型将是一个很有效的工具。     

藻红蛋白在微乳液中的增溶溶解

本文研究了藻红蛋白在油包水微乳液的紫外吸收和荧光光谱,结果表明,含藻红蛋白的阳离子表面活性剂的微乳液的光谱与其水溶液的光谱不同,说明藻红蛋白在这样的微环境中变性,与此相反,含藻红蛋白的阴离子表面活性剂的微乳液的光谱与其水溶液的相应光谱的特征峰相同,说明藻红蛋白在阴离子表面活性剂的微乳液中没有变性。本文进一步研究了含藻红蛋白的阴离子表面活性剂的微乳液在不同含水量时蛋白质的稳定性。并根据蛋白质的大小和     

Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function.

Mutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp-26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10-fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp-26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro-34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro-76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.     

Determination of intoplicine, a new antitumour drug, in human whole blood and plasma by normal-phase high-performance liquid chromatography with fluorescence detection

Intoplicine, a benzopyrido-indole derivative, is a novel anticancer agent currently under phase I clinical evaluation. A selective, sensitive normal-phase high-performance liquid chromatographic (HPLC) assay with fluorescence detection, suitable for the determination of intoplicine in human plasma and whole blood, is described. The sample pretreatment involves a protein precipitation step with 2-propanol. The reported assay was validated, and the stability of the analyte in plasma, in whole blood and in the extraction fluid was investigated. The method has been implemented in a pharmacokinetic phase I clinical trial with intoplicine given as a 24-h intravenous infusion.     

The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK8 greater than CCK33 greater than desulphated CCK8 greater than CCK4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [125I]CCK was cross-linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with Mr 80 000 protein in both the presence and absence of the reducing agent dithiothreitol.     

Modern-day challenges in therapeutic protein production

The market for therapeutic proteins is on the rise, plagued by several challenges related to production amounts and costs. Solutions to these problems are widely thought to come from academia, governments and production companies. This conference aimed to bring experts in the industry together under one roof, in order to demystify several novel technologies in therapeutic protein development. Key topics included analytical tools for protein stability and ligand interactions, measurement of protein aggregates as small as 30 nm and reducing production costs, just to name a few. The need to eliminate protein aggregates early during bioprocessing was emphasized. Finally, several companies presented novel technologies related to therapeutic protein development.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

ODN 491, a novel antisense oligodeoxynucleotide that targets thymidylate synthase, exerts cell-specific effects in human tumor cell lines

Thymidylate synthase (TS) is essential for DNA replication and is a target for cancer chemotherapy. However, toxicity to normal cells and tumor cell drug resistance necessitate development of new therapeutic strategies. One such strategy is to use antisense (AS) technology to reduce TS mRNA and protein levels in treated cells. We have developed oligodeoxynucleotides (ODNs) that target different regions of TS mRNA, inhibit human tumor cell proliferation as single agents, and enhance cytotoxicity of clinically useful TS protein-targeting drugs. Here we describe ODN 491, a novel 20mer AS ODN complementary to a previously untargeted portion of the TS mRNA coding region. AS ODN 491 decreased TS mRNA levels to different degrees in a panel of human tumor-derived cell lines, and induced different physiological effects in a tumor cell line-dependent manner. ODN 491 (like AS TS ODN 83, previously shown to be effective) decreased TS protein levels in HeLa cells with a concomitant increase in sensitivity to TS-targeting chemotherapeutics. However (and contrary to HeLa cell response to an AS ODN 83), it did not, as a single agent, inhibit HeLa cell proliferation. In MCF-7 cells, ODN 491 treatment was less effective at reducing TS mRNA and did not reduce TS protein, nor did it enhance sensitivity to TS-targeting or other chemotherapeutics. Moreover, specifically in MCF-7 cells but not HeLa cells, ODN 491 as a single agent induced apoptosis. These data indicate that AS TS ODN 491 is an effective AS reagent targeting a novel TS mRNA region. However, treatment of tumor cell lines with AS TS ODNs targeting different TS mRNA regions results in a pattern of physiological effects that varies in a tumor cell line-specific fashion. In addition, the capacity of different AS TS ODNs to induce physiological effects does not correlate well with their capacity to reduce TS mRNA and/or protein and, further, depends on the region of TS mRNA selected for targeting. Recognition of tumor cell-specific and mRNA region-specific variability in response to AS TS ODNs will be important in designing AS TS ODNs for potential clinical use.     

Cysteine-free mutant of aequorin as a photolabel in immunoassay development

The bioluminescent protein aequorin is a sensitive label that has been employed in a number of analytical applications. A mutant of aequorin with enhanced stability produced recombinantly in our laboratory has been employed as a label in the development of an immunoassay for digoxin. Digoxin is a cardiac glycoside used in the treatment of congestive heart failure. This drug has a very narrow therapeutic range of 0.8-2.0 ng/mL (1.0-2.5 nmol/L), thus requiring therapeutic drug monitoring. In this study, a derivative of digoxigenin was chemically conjugated to the mutant aequorin, and the resulting protein-digoxigenin derivative conjugates were characterized in terms of their luminescence properties. A solid-phase immunoassay for digoxin was then developed. The detection limit of the assay for digoxin was 1 x 10(-12) M. To demonstrate the use of this mutant aequorin as a label in biological sample analysis without any need for pretreatment of the samples, the assay was tested in serum spiked with digoxin. Interference from digoxin analogues was also evaluated to determine the specificity of the assay.     

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.     

Leukotriene synthesis in response to A23187 is inhibited by methyl- beta-cyclodextrin in RBL-2H3 cells

Leukotrienes (LTs) are produced by several biosynthetic enzymes including cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase activating protein (FLAP) in the perinuclear area. In the present study, we showed that pretreatment with methyl-beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent, dramatically reduced the synthesis of LTs in response to A23187 in mast cells. A23187-induced LT synthesis was inhibited by pretreatment with MbetaCD, and this effect was reversed when cholesterol was added. In an approach to identifying the MbetaCD-sensitive protein(s), we observed that FLAP co-localized with flotillin-1, a lipid raft marker protein, in the lipid raft-rich low-density region of sucrose gradients. In addition, electron microscopic analysis revealed that FLAP co-localized with flotillin-1. Together, these results suggest that FLAP is present in cholesterol-rich lipid raft-like domains and that its localization in these domains is critical for LT synthesis.