生物传感芯片质谱及其在蛋白质组研究中的应用

目前蛋白质组研究技术在已有的研究基础上取得了长足进展 ,包括固相 ｐＨ梯度双向凝胶电泳、生物质谱技术、蛋白质双向电泳图谱的数字化和各种分析技术、蛋白质间相互作用分析的方法如酵母双杂交系统、噬菌体展示、表面等离子共振等。但均各有其利弊。目前质谱是进行蛋白质组成分鉴定的支撑技术 ,但是它在蛋白质间相互作用分析及结构与功能的关系分析方面显得无能为力 ,而生物分子相互作用分析技术 (Ｂｉｏｍｏｌｅｃｕｌａｒｉｎｔｅｒａｃｔｉｏｎａｎａｌｙｓｉｓ ,ＢＩＡ)是进行蛋白质相互作用分析的较好技术 ,将两者结合起来在蛋白质组…     

酵母双杂合系统的改进和发展

酵母双杂合系统是在１９８９年由ＳｔａｎｌｅｙＦｉｅｌｄｓ和Ｏｋ－ｋｙｕＳｏｎｇ等提出并初步建立的［１］,该系统是在酿酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）中研究蛋白质间相互作用的一种非常有效的分子生物学方法。近几年来随着人们对该系统的广泛应用,这一系统得到了不断的完善及改进,同时也衍生出单杂合系统,三杂合系统等一系列相关的技术。这些技术在不同研究领域中的广泛应用有力地推动了蛋白质与ＤＮＡ,蛋白质与ＲＮＡ,以及多种蛋白质分子间相互作用的研究。     

新书介绍

《ＤＮＡ 蛋白质相互作用》(ＤＮＡ ＰｒｏｔｅｉｎＩｎｔｅｒａｃｔｉｏｎｓ)ＡｎｄｒｅｗＴｒａｖｅｒｓ和ｍａｌｃｏｌｍＢｕｃｋｌｅ编著 ,2 0 0 0年ＯｘｆｏｒｄＵｎｉｖｅｒｓｉｔｙＰｒｅｓｓ出版 ,375页。蛋白质与ＤＮＡ的相互作用在分子生物学领域占有重要地位。例如 ,在确定和调节染色体结构、ＤＮＡ复制、重组和修复、基因转录、病毒感染等方面具有很大作用。本书是研究蛋白质与ＤＮＡ相互之间特异作用及许多研究方法的概述。描述了目前适用的许多主要的技术方法 ,尤其是分析转录及它与染色质结构关系的生物物理方法。每一种方法…     

噬菌体展示肽库在细胞信号转导研究中的应用

细胞信号转导是一个由信号分子相互作用介导的生物信息传递的过程 ,蛋白质之间以及蛋白质与其他分子间的相互作用是信号转导的基础。近年来 ,利用噬菌体展示肽库技术研究细胞信号转导 ,取得了很多有意义的结果。1.鉴定信号蛋白识别结合位点信号蛋白之间特异性的识别结合主要通过一小段保守区实现 ,如ＳＨ2区 (Ｓｒｃ ｈｏｍｏｌｏｇｙｄｏｍａｉｎ)、ＳＨ3区、ＰＴＢ区 (ｐｈｏｓｐｈｏｔｙｒｏｓｉｎｅ ｂｉｎｄｉｎｇｄｏｍａｉｎ)等。用噬菌体展示肽库技术可以快速获得与这些保守区结合的配体的结构信息。用Ｓｒｃ酪氨酸激酶的ＳＨ3区筛…     

线粒体与细胞凋亡机制

细胞凋亡是生理性的细胞死亡过程,受到多种基因的精确调节,一类被统称为ｃａｓｐａｓｅ的半胱氨酸蛋白酶是细胞凋亡程序的执行者,综们被激活后作用于细胞内的一些蛋白质,经起细胞凋亡。线粒体中含有许多凋亡相关因子,在凋亡信号转导中起着重要作用。细胞受到凋亡刺激后,细胞色素ｃ、ＡＩＦ、ｃａｓｐａｓｅ－９等凋亡相关因子从线粒体中释放出来。细胞色素ｃ通过和Ａｐａｆ－１、ｃａｓｐａｓｅ－９相互作用,激活ｃａｓｐａｓ     

菜豆富含脯氨酸蛋白质基因在生物和非生物胁迫下的表达

植物细胞壁是细胞的机械支持物和保护壳,在植物的生长和发育、细胞内的通讯和代谢交换过程中起重要作用。细胞壁主要由多糖和蛋白质组成,根据蛋白质的氨基酸组成和糖的含量特点可把细胞壁结构蛋白分为５类：富含羟脯氨酸的糖蛋白（ｂｙ一ｄｒｏｘｙｐｒｏｌｉｎｇ－ｒｉｃｈ　ｇｌｙｃｏｐｒｏｔｅｉｎｓ,ＨＲＧＰｓ）、富含脯氨酸蛋白质（ｐｒｏｌｉｎｅ－ｒｉｃｈｐｒｏｒｅｉｎｓ,ＰＲＰｓ）、富含甘氨酸蛋白质（ｇｌｙｃｉｎｅ－ｒｉｃｈｐｒｏｔｅｉｎｓ,ＧＲＰｓ）、茄科凝集素（ｓｏｌａｎａｃｅｏｕｓｌｅｃｔｉｎｓ）和阿拉伯…     

APC基因结构及功能异常与人类肿瘤的关系

腺瘤样结肠息肉病易感基因是１９９１年从结肠癌中克隆到的一个新的抑癌基因,定位于５号染色体长臂。ＡＰＣ基因产物功能不茂清楚,发现其可调节ｃ－ｍｙｃ基因ｍＲＮＡ的成熟和ｒａｓ基因的表达,与蛋白质相互作用有关。     

酵母以杂合系统的改进和发展

酵母双杂合系统是在１９８９年由ＳｔａｎｌｅｙＦｉｅｌｄｓ和Ｏｋ－ｋｙｕ Ｓｏｎｇ等提出并初步建立的＾［１］,该系统是在酿酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）中研究蛋白质间相互作用的一种非常有效的分子生物学方法。近几年来随着人们对该系统的广泛应用,这一系统得到了不断常有的效的分子生物方法。近几年业随着人们对该系统的广泛应用,这一系统得到了不断的完善及改进,同时也衍生出单杂合系统     

逆向酵母双杂交系统的应用

198 9年Ｆｉｅｌｄｓ等提出了酵母双杂交系统来研究真核细胞的蛋白质之间的相互作用。这一崭新手段迅速发展成为一种常规的分子生物学技术 ,并得到了广泛的应用。近来 ,国外学者为拓宽传统的双杂交系统的应用范围 ,在原有的酵母双杂交的基础上发展了大量衍生系统 ,包括用于研究蛋白质与ＤＮＡ之间相互作用的单杂交系统 (ｏｎｅ ｈｙｂｒｉｄｓｙｓ ｔｅｍ) [1] ;研究两个蛋白质与第三个成分间相互作用的三杂交系统 (ｔｈｒｅｅ ｈｙｂｒｉｄｓｙｓｔｅｍ) [2 ] ,第三个成分可以是蛋白质、ＲＮＡ或小分子药物等[3 ] 。另外 ,在通过双杂交体系…     

木质素过氧化物酶基因5'端上游调控序列的分析

黄孢原毛平革菌（Ｐｈａｎｅｒｏｃｈａｅｔｅｃｈｒｙｓｏｓｐｏｒｉｕｍ）能产生降解木质素的胞外木质素过氧化物酶（ＬＩＰ） 和锰过氧化物酶（ ＭｎＰ）同工酶。为研究ＬＩＰ基因的转录调控机理, 对ＬＩＰ基因（ ＧＬＧ３ 和ＧＬＧ６） 的５′端上游序列进行亚克隆, 获得６ 个亚克隆ＤＮＡ 片段, 然后应用凝胶迁移率变动分析技术筛选能与菌体蛋白质专一性结合的ＤＮＡ片段。结果表明： ＬＩＰ基因ＧＬＧ６ 的５′端上游有一个约６７０ ｂｐ 的ＤＮＡ 片段能与总蛋白质组分专一性结合, 其核苷酸序列分析表明该片段可能含有蛋白质结合的序列特征。研究结果初步显示, 黄孢原毛平革菌可能存在有与ＬＩＰ基因上游某些顺式调控元件相互作用的蛋白质, 调控着ＬＩＰ基因的转录表达。     

Protein-protein interactions: methods for detection and analysis.

The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques.     

适配体结合蛋白质靶标的亲和力表征方法及其在疾病诊断中的应用

核酸适配体是通过体外指数富集配体系统进化（SELEX）技术筛选获得,并能够和蛋白质靶标高特异性、高亲和力结合的单链寡核苷酸。核酸适配体不但具有抗体的识别特性,而且具有自己独特的优良性能,目前已应用于分析检验、食品安全和生物医药等各个领域。蛋白质具有多种多样的生物功能以及临床诊断价值。因此,核酸适配体针对蛋白质靶标并在蛋白质相关的基础研究领域受到广泛的关注。核酸适配体应用性能的优劣取决于与其靶标蛋白质的亲和力与特异性。本文主要综述核酸适配体对蛋白质靶标的亲和力表征方法,以及在药物研发、肿瘤检测、生物成像以及生物传感器方面的应用。     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

New Frontiers for Machine Learning in Protein Science

Protein function is fundamentally reliant on inter-molecular interactions that underpin the ability of proteins to form complexes driving biological processes in living cells. Increasingly, such interactions are recognised as being formed between proteins that exist on a broad spectrum of dynamic conformational states and levels of intrinsic disorder. Additionally, the sizes of the structures formed can range from simple binary complexes to large dynamic biomolecular condensates measuring 100 nm or more. Understanding the parameters that govern such interactions, how they form, how they lead to function and what happens when they take place in unintended manners and lead to disease, represent some of the core questions for molecular biosciences. In light of recent advances made in solving the protein folding problem by machine learning methods, we discuss here the challenges and opportunities brought by these new data-driven approaches for the next frontiers of biomolecular science.     

Protein microarrays: promising tools for proteomic research

Miniaturized and parallelized ligand binding assays are of great interest in postgenomic research because microarray technology allows the simultaneous determination of a large number of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are used for the identification and quantification of proteins as well as for the study of protein interactions. Protein affinity assays have been implemented that allow the analysis of interactions between proteins with other proteins, peptides, low molecular weight compounds, oligosaccharides or DNA. Microarray technology is an emerging technology used in global analytical approaches and has a considerable impact on proteomic research.     

Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

A major focus of systems biology is to characterize interactions between cellular components, in order to develop an accurate picture of the intricate networks within biological systems. Over the past decade, protein microarrays have greatly contributed to advances in proteomics and are becoming an important platform for systems biology. Protein microarrays are highly flexible, ranging from large-scale proteome microarrays to smaller customizable microarrays, making the technology amenable for detection of a broad spectrum of biochemical properties of proteins. In this article, we will focus on the numerous studies that have utilized protein microarrays to reconstruct biological networks including protein-DNA interactions, posttranslational protein modifications (PTMs), lectin-glycan recognition, pathogen-host interactions and hierarchical signaling cascades. The diversity in applications allows for integration of interaction data from numerous molecular classes and cellular states, providing insight into the structure of complex biological systems. We will also discuss emerging applications and future directions of protein microarray technology in the global frontier.     

Alkynyl-farnesol reporters for detection of protein S-prenylation in cells

Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.     

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.     

Protein interaction networks in bacteria

The complement of expressed cellular proteins - the proteome - is organized into functional, structured networks of protein interactions that mediate assembly of molecular machines and dynamic cellular pathways. Recent studies reveal the biological roles of protein interactions in bacteriophage T7 and Helicobacter pylori, and new methods allow to compare and to predict interaction networks in other species. Smaller scale networks provide biological insights into DNA replication and chromosome dynamics in Bacillus subtilis and Archeoglobus fulgidus, and into the assembly of multiprotein complexes such as the type IV secretion system of Agrobacterium tumefaciens, and the cell division machinery of Escherichia coli. Genome-wide interaction networks in several species are needed to obtain a biologically meaningful view of the higher order organization of the proteome in bacteria.     

Understanding Protein Mobility in Bacteria by Tracking Single Molecules

Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm.