该项目是支持部分由Alfred P. Sloan基金会NSF事业奖（DBI 1027394），和国家普通医学科学研究所（GM083107，GM084222）。

<ol>
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Chem. 25, 865-871 (2004).</li><li>Zhang, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+protein+structure+refinement+using+fragment+guided+molecular+dynamics+simulations.">High-resolution protein structure refinement using fragment guided molecular dynamics simulations.</a> , (2010).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-TASSER+server+for+protein+3D+structure+prediction.">I-TASSER server for protein 3D structure prediction.</a> BMC Bioinformatics. 9, 40-40 (2008).</li><li>Xu, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+significant+is+a+protein+structure+similarity+with+TM-score+=+0.5?.">How significant is a protein structure similarity with TM-score = 0.5?.</a> Bioinformatics. 26, 889-895 (2010).</li><li>Zhang, Y., Skolnick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scoring+function+for+automated+assessment+of+protein+structure+template+quality.">Scoring function for automated assessment of protein structure template quality.</a> Proteins. 57, 702-710 (2004).</li><li>Barrett, A. 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没有利益冲突的声明。

上述协议是一个使用我的TASSER服务器的结构和功能建模的一般准则。虽然，这种自动化程序非常适用于大多数的蛋白质，人类的干预往往有助于显著提高建模精度，尤其是蛋白质缺乏在PDB库的密切模板。用户可以干预在我TASSER建模在以下方面：（一）多结构域蛋白的分裂;（b）提供外部约束，以改善结构组装;及（c）消除在建模的模板。 分裂多结构域蛋白： 许多长期的蛋白质序列通常包含多个域，灵活的连接器的地区，这使得其结构鉴定难以用实验和计算技术拴。然而，作为域是独立折叠实体，并可以执行不同的分子功能;宜分裂长的多结构域蛋白和模型，每个域分开。建模领域单独将不仅加快了预测过程，但也增加了查询模板对齐的质量，更可靠的结构和功能预测。 可以预见，在蛋白质序列域边界，使用免费提供的外部网上课程，如NCBI 的 CDD 24 PFAM 25或26 InterProScan。此外，如果LOMETS线程路线可供查询的蛋白，可域边界位于视觉识别很长一段未对齐的残留在顶部的线程模板（见第5.4步）。这些未对齐的地区，大多对应域连接器区域。如果多域模板已经在模板中的所有查询对齐域的PDB库，然后查询蛋白可以仿照全长。 提供外部约束<p类=“jove_content”&gt;在我的TASSER结构装配模拟，主要是从线程模板LOMETS收集的空间限制的指导。有良好的线程命中（Norm. Z - score模型&gt; 1）在模板库查询蛋白质，派生的空间限制，大多精度高，我TASSER将这些蛋白质生成高分辨率的结构模型。相反，查询的蛋白质，有弱或没有线程命中（Norm. Z分数 &lt;1），收集了空间的限制，往往含有错误，因为模板对齐的不确定性。对于这些蛋白质的目标，用户指定的空间信息，可以提高预测模型的质量是非常有益的。用户可以提供两种方式我TASSER服务器的外部约束： A.指定联系人/距离限制 实验特点的跨残留的接触/距离，例如从核磁共振交叉连接实验，可以通过上传约束文件指定。一个例子文件是在图8所示，其中第1列指定类型的约束，即“距离”或“联系”。对于距离克制（测距），2和第4列含有残留物的位置（I，J），3和第5列中的残留物和第6列中包含的原子类型指定两个指定的原子之间的距离。对于接触限制（接触），2和第3列包含的残留量应在接触的位置（I，J）。这些联系的残留物对侧链中心之间的距离决定的基础上已知的PDB结构的观察距离。我TASSER将尝试绘制这些原子对指定的距离在结构细化模拟。 B.指定蛋白质的结构模板 LOMETS线程程序使用有代表性的PDB库，寻找可能的查询PROT褶皱EIN。虽然使用有代表性的结构库，有助于减少计算的序列结构路线所需的时间，它是可能错过一个很好的模板蛋白是在图书馆或模板可能没有被LOMETS线程方案确定，即使它是目前在库中。在这种情况下，用户应指定为模板所需的蛋白质的结构。 要指定一个额外的模板的蛋白质结构，用户可以上传PDB格式的结构文件，或指定在PDB库中存放的蛋白质结构的PDB ID。我的TASSER将生成的查询模板对齐， 使用鼓起方案23，将收集指定用户的模板和LOMETS模板，引导结构装配仿真空间的限制。由于LOMETS限制的准确性是针对不同的目标不同，LOMETS限制重量是容易（同源）TArgets比在硬盘（非同源）的目标，这已经在我们的基准培训系统调整。 用户还可以指定自己的查询模板路线。服务器接受对齐两种格式：FASTA格式（图9A）和3D格式（图9b）。 FASTA格式的标准和描述<a href="http://zhanglab.%20ccmb.med.umich.edu/FASTA/">http://zhanglab。 ccmb.med.umich.edu / FASTA /</a> 。 3D格式是类似的标准PDB格式（ <a href="http://www.wwpdb.org/documentation/format32/sect9.html">http://www.wwpdb.org/documentation/format32/sect9.html</a> ），但来自模板的两个附加列被添加到原子记录（见图9B）： 列1-30：原子（C -α）和残留查询序列的名称。 列31-54：从模板中的相应原子复制查询的C -α的原子坐标。 列55-59：在相应的模板残留数量的基础上对齐 列60-64：通讯残留在模板名称排除模板蛋白质蛋白质是灵活的分子，可以采取多种构象状态，以改变其生物活性。例如，许多蛋白激酶和膜蛋白的结构已经解决了active 和 inactive的构象。约束配体的存在或缺乏可造成大的构造运动。虽然所有的构象状态的模板是线程程序一样，它是可取的模式查询使用模板，只在一个特定的状态。在服务器上的一个新的选项允许用户排除在结构造型的模板蛋白。此功能也允许用户选择的造型模板的同源性水平。用户可以排除模板蛋白FROM我TASSER库： A.指定序列的身份截止 用户可以使用这个选项排除我TASSER模板库的同源蛋白。同源性级别设置基于序列的身份截止，即除以查询顺序序列的长度之间的查询和模板蛋白相同的残渣的数量。例如，如果用户的类型，在“70％”，在所提供的形式，所有模板蛋白质&gt; 70％，我将我TASSER模板库中排除查询蛋白质序列的身份。 B.排除特定的模板蛋白上传一个列表，其中包含要排除的结构的PDB的ID，可以排除特定的模板蛋白的I - TASSER模板库。图10显示了一个例子文件。由于相同的蛋白质可以存在多个条目，在PDB库，我TASSER SErver默认情况下，将排除指定的模板（Column1中）以及从库中，有一个身份的所有其他模板&gt; 90％，到指定的模板。用户还可以指定一个不同的身份截止，如70％，所有与身份模板&gt; 70％，到指定的模板蛋白将被排除在外。

<ol><li> FASTA格式的氨基酸序列为蓝本的蛋白质（ <a href="http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml">见</a> http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml） 。 </li><li>与个人电脑接入互联网和Web浏览器。 </li><li>分子可视化软件，如RASMOL或PYMOL，分析预测的三级结构和功能的网站。 </li></ol>

a protocol for computer-based protein structure and function prediction

基因组测序项目，以加密形式以百万计的蛋白质序列，这就要求它们的结构和功能的知识，提高其生物作用的认识。虽然实验方法提供了这些蛋白质的一小部分的详细信息，计算模型所需要的大多数实验未知的蛋白质分子。我TASSER服务器是一个高分辨率的蛋白质结构与功能的建模上线的工作台。由于蛋白质序列，从我的TASSER服务器上的一个典型输出包括二级结构预测，预测每个残基的溶剂可及，同源模板线程和结构对齐检测蛋白质，最多五个全长高等教育的结构模型，和基于结构酶的分类，功能注释基因本体术语和蛋白质 - 配体结合位点。所有的预测标记的信心分数讲述如何准确的预测是不知道的实验数据。为了方便最终用户的特殊要求，的服务器提供渠道接受用户指定的残留间的距离和联系映射到交互地改变我TASSER建模，而且还允许用户指定的任何蛋白质为模板，或排除任何模板蛋白质在结构装配模拟。结构信息可以收集用户基于实验证据或生物的见解与我TASSER预测的质量改善的目的。服务器被评为蛋白质的结构和功能预测，在最近社会广泛CASP实验的最佳方案。目前有20000注册来自100多个国家的科学家使用的是上线的我，TASSER服务器。

Watch this Scientific Journal Video about A Protocol for Computer-Based Protein Structure and Function Prediction at JoVE.com

A Protocol for Computer-Based Protein Structure and Function Prediction

计算机准则的基础结构和功能的蛋白质使用的I - TASSER管道的特性描述。开始，从查询蛋白质序列生成三维模型使用多个线程的路线和迭代结构装配模拟。此后绘制功能的推论是基于匹配与已知的结构和功能的蛋白质。

基于计算机的蛋白质结构与功能预测议定书

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

a-protocol-for-computer-based-protein-structure-function

Research

JoVE Journal

Biology

68.1K 次观看.  University of Michigan. 计算机准则的基础结构和功能的蛋白质使用的I - TASSER管道的特性描述。开始，从查询蛋白质序列生成三维模型使用多个线程的路线和迭代结构装配模拟。此后绘制功能的推论是基于匹配与已知的结构和功能的蛋白质。

视频: A Protocol for Computer-Based Protein Structure and Function Prediction

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

发现蛋白质相互作用和检定蛋白质功能使用的HaloTag技术

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

诱导LAP标记的稳定细胞系用于研究蛋白质功能，时空定位与蛋白质相互作用网络

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this ...