箭毒木种子蛋白质样品制备及双向电泳改良方法

建立箭毒木（Antiaris toxicaria）种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris—HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris—HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris—HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。     

莲种子蛋白质提取方法比较与双向电泳分析

莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。     

玉米花丝蛋白质组双向电泳条件的优化

以玉米温敏自交不亲和系‘HE97’的花丝为材料,比较了3种不同蛋白质提取方法对双向电泳结果的影响,并对其中的蛋白质上样量、等电聚焦条件及SDS-PAGE凝胶浓度进行了探索与优化。结果表明,与酚提取法和改良的酚提取法相比,采用三氯乙酸/丙酮提取法提取蛋白质操作简便,所得的双向电泳图谱蛋白质点数较多,图谱背景清晰,是一种提取玉米花丝蛋白质的有效方法。优化后的双向电泳技术体系适合于玉米花丝全蛋白质的双向电泳分析。     

油菜叶片总蛋白质双向电泳样品制备方法的改进

以甘蓝型油菜"扬油6号"的叶片为试验材料,分别采用传统的TCA/Acetone(三氯乙酸/丙酮沉淀法)和改进的PEG(polyethylene glycol)分步提取法提取叶片可溶性总蛋白,并利用条件一致的蛋白质双向电泳体系进行比较。TCA/Acetone法提取的蛋白质双向电泳图谱背景中由于高丰度"housekeeping"结构蛋白的存在,特别是叶片中参与光合作用的Rubisco蛋白的干扰,图谱中低丰度调控蛋白受到了高度覆盖和遮蔽现象,影响双向电泳图谱的质量。而PEG分步提取法提取的蛋白质样品,可以剔除Rubisco蛋白,使获得的双向电泳图谱清晰,无斑点间的遮蔽现象,为油菜叶片蛋白质组定量和定性分析提供了丰富的信息。     

砂藓总蛋白质提取方法的选取及双向电泳体系的建立

为进行砂藓(Racomitrium canescens)蛋白质组学的双向电泳工作,首要前提是获得质量较好的砂藓总蛋白质,并建立一套完整高效的适用于砂藓的双向电泳体系。本研究以砂藓为研究对象,比较了Tris-酚抽提法、TCA-丙酮法和试剂盒法3种蛋白质提取方法获得砂藓总蛋白质的浓度和完整性,并对砂藓中蛋白质双向电泳的运行条件进行了优化和比较。结果表明:在3种砂藓总蛋白质提取方法中,Tris-酚抽提法获得的砂藓总蛋白质浓度最高,SDS-PAGE电泳结果中条带清晰且较完整,双向电泳后获得的蛋白质点最多,是3种方法中提取砂藓蛋白质浓度最高完整性最好的方法;基于Tris-酚抽提法的基础上进一步对砂藓总蛋白质双向电泳体系进行优化,结果显示:利用pH值范围为pH 4～7的IPG胶条,蛋白质上样量为1 000μg的条件下,获得的砂藓蛋白质双向电泳图谱蛋白质点最多,图谱中可分辨的蛋白质点共(872±15)个,且低丰度蛋白质点清晰。本研究筛选出的方法获得了蛋白质点分布均匀、背景清晰、分辨率高、质量较高的双向电泳图像,建立了砂藓蛋白质组学研究体系的较好方法。本研究结果为砂藓蛋白质组学研究提供了良好基础。     

粘虫中肠总蛋白质双向电泳体系的优化方法

为了今后更好的利用双向电泳技术研究不同处理后粘虫Mythimna separata中肠蛋白表达差异,本研究比较了不同的蛋白提取方法、IPG胶条、二硫苏糖醇(DTT)浓度和等电聚焦条件,建立了适用于粘虫中肠总蛋白质的双向电泳体系。结果表明:采用Tris-饱和酚法提取蛋白质,选取p H为5-8的线性IPG胶条进行第一向等电点分离,按照DTT浓度I和等电聚焦程序II进行双向电泳分离,获得了蛋白质点数多、背景清晰、分辨率高的蛋白质双向电泳图谱。     

HBsAg生化性状对乙型肝炎疫苗效力影响的研究

比较研究了重组酵母HBsAg P24亚基间二硫键结合及其还原条件下降解程度对乙型肝炎疫苗效力的影响。分别用HPLC及SDS－PAGE法检测重组酵母乙型肝炎疫苗HBsAg的P24亚基在还原条件下降解和P24亚基间二硫键桥联程度,并检测乙型肝炎疫苗小鼠体内效力（ED50值）和体外相对效力（RP）,分析HBsAg的P24亚基间二硫键结合及其还原条件下降解程度对乙型肝炎疫苗效力的影响。结果表明HBsAgP24亚基还原条件下降解成分Sub-P24的相对含量在5．2％－31％间,P24亚基间二硫键桥联型HBsAg颗粒相对含量在41．9％－71．8％范围内与疫苗的实验室效力无关。所生产重组酵母乙型肝炎疫苗HBsAg的P24亚基间二硫键结合及其还原条件下降解程度不影响乙型肝炎疫苗实验室效力。     

蓝色温和凝胶双向电泳用于分析嗜盐菌质膜蛋白复合物

为了揭示细胞对盐胁迫渗透适应的分子机制,以新鉴定的中度嗜盐芽孢杆菌Bacillussp.I121为实验材料,分析了该嗜盐菌质膜上的盐胁迫响应蛋白.为此,通过蓝色温和凝胶双向电泳(BN/SDS-PAGE)对纯化的质膜组分进行了差异蛋白质组学研究.经MALDI-TOF/TOF质谱分析,鉴定了8个盐胁迫响应蛋白.盐胁迫诱导上调表达的蛋白质包括ABC型转运蛋白、3-磷酸甘油透性酶、嘧啶核苷转运蛋白和甲酸脱氢酶,下调表达的蛋白质包括琥珀酸脱氢酶(succinate dehydrogenase)铁硫亚基、黄素蛋白亚基、细胞色素b556亚基以及分子伴侣DnaJ的同源蛋白;酶活力测定结果表明胁迫条件下上述蛋白质的活性变化与表达量变化相一致.这些蛋白质中绝大多数属于高度疏水的跨膜蛋白,主要负责物质跨膜运输及能量代谢.上述结果表明,中度嗜盐菌Bacillus sp.I121可通过加快跨膜物质运输,同时抑制TCA循环完成盐胁迫条件下相容性溶质脯氨酸和四氢嘧啶的合成与积累.也进一步证明,蓝色温和凝胶双向电泳不仅可用于线粒体、叶绿体中蛋白质复合物的分析,也同样适用于细胞质膜上高度疏水蛋白复合物的比较研究.     

双向电泳分析鸢尾绿白嵌合叶片的蛋白质

利用双向聚丙烯酰胺凝胶电泳对鸢尾（Iris japonica)绿白嵌合叶片的蛋白质进行分离,并初步鉴定了蛋白质的相对分子量和等电点。每个电泳图谱共检测到400余个蛋白点,其中至少13个蛋白的表达变化明显;结果表明,嵌合叶片的绿色与白色叶组织具有明显不同的蛋白质双向电泳图谱。与数据库中拟南芥双向电泳图谱相比较,发现Rubisco大亚基,标记为W和T蛋白的表达变化与产生绿白嵌合叶片的表型密切相关。     

红苋P104种子谷蛋白的电泳分析

通过单向不平变性聚丙烯酰胺凝胶等电聚焦（ＩＥＦ）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）和双向电泳（ＩＥＦ×ＳＤＳ－ＰＡＧＥ）分析了红苋Ｒ１０４种子谷蛋白的亚基组成,亚其分子量和等电点分布。谷蛋白的双向电泳图谱可分辨出１００多个亚基成分,其主要亚基为：５４ｋＤ（ｐＩ７．１５）;３３ｋＤ（ｐＩ５．８２）;３１ｋＤ（ｐＩ６．９２;ｐＩ６．７０;ｐＩ６．６５）;２２ｋＤ（ｐＩ８．３４）;２     

高分子量麦谷蛋白１４和１５亚基的纯化、Ｎ—末端序列及部分生化特性研究

用ＳＤＳ－ＰＡＧＥ制备电泳技术结合一种新的凝胶中蛋白质显色方法,对普通小麦（Triticum aestivum)小偃六号的高分子量麦谷蛋白１４和１５亚基进行了有效的分离纯化,将其转印于ＰＶＤＦ膜上测定了Ｎ－端的氨基酸顺序,通过比较了发现它们与已知序列的其他的高分子是麦谷蛋白亚基高度同源。用两种双向电泳技术确定了它们的等电点（ＰＩ）属于碱性范围。     

Kinetics of ribosome synthesis during a nutritional shift-up in Escherischia coli K-12.

Summary The rates of total protein synthesis, polyribosome formation and 70S ribosome accumulation were measured following a nutritional shift-up ofEscherichia coli K-12. Changes in ribosome content and distribution during the shift-up were measured by examining the total cellular content of free and polysome-associated ribosomes using a sensitive double isotope labeling method. The kinetics of ribosomal subunit formation and the biosynthesis of subunit protein and RNA species were also defined. The results indicated that a pre-shift population of ribosomal subunits was utilized for the immediate post shift increase in both total and ribosomal-specific protein synthesis. An assembly time for new subunits of about 3 min was observed. The formation of certain ribosomal proteins during the shift suggested that new subunit assembly was limited by the rate of synthesis of particular ribosomal proteins during this growth transition.     

A simplified method for reconstituting active E. coli DNA polymerase III

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ?, θ, γ, δ′, δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be co-expressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.     

Isolation of thylakoid membrane complexes from rice by a new double-strips BN/SDS-PAGE and bioinformatics prediction of stromal ridge subunits interaction

Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new "Double-Strips BN/SDS-PAGE" method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Effect of acetic acid on phycocyanin-phycoerythrocyanin mixture extracted from Anabaena variabilis

Exposure of a phycocyanin-phycoerythrocyanin mixture extracted from Anabaena variabilis to sodium acetate, pH 3.8, ionic strength of 0.1, results in dissociation of the phycoerythrocyanin's beta subunit from its alpha subunit. The alpha subunit obtained by this method has a strong absorption transition at 508 nm. This transition is a consequence of the subunit's specific conformation, rather than of a new chromophore. The behavior of the phycocyanin-phycoerythrocyanin mixture in acetate buffers of variable compositions suggests that interactions which involve carboxylic amino acid residues play an important role, along with hydrophobic associations, in the association of phycoerythrocyanin subunits into monomers (alpha beta) and between this protein and phycocyanin. This work also indicates that the linkage between alpha and beta subunits of phycoerythrocyanin is labile and may be weaker than the association of these subunits with phycocyanin under acidic conditions.     

InterProSurf: a web server for predicting interacting sites on protein surfaces

A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. AVAILABILITY: The InterProSurf web server is available at http://curie.utmb.edu/     

Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins

Streptococcus suis serotype 2 (SS2) is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP) and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.