プロジェクトは、アルフレッドP.スローン財団、NSFのキャリア賞（DBI 1027394）、および米国立総合医科学研究所（GM083107、GM084222）によって部分的にサポートされています。

<ol>
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利害の衝突は宣言されません。

上記のプロトコルは、I - TASSERサーバを使用して構造と機能のモデリングのための一般的なガイドラインです。 、この自動化された手順は、タンパク質の大部分を非常にうまく動作しますが、人間の介入は、しばしば特にPDBライブラリに近いテンプレートを欠いているタンパク質のため、大幅にモデリングの精度を向上させるのに役立ちます。 （b）の構造のアセンブリを改善するための外部制約を提供する;マルチドメインタンパク質の（A）の分割及び（c）のモデル化の際にテンプレートを削除：ユーザーは、次の方法でI - TASSERのモデリング中に介入することができます。 分割するマルチドメイン蛋白質： 多くの長いタンパク質配列は、頻繁に実験と計算の両方の技術を使用して、その構造の解明を困難にする柔軟なリンカー領域によってつなが複数のドメインが含まれています。それにもかかわらず、ドメインは独立して折り畳み式の実体であり、明確な分子機能を実行することができますように、それは個別に各ドメインの長いマルチドメインタンパク質とモデルを分割することが望ましい。モデリングのドメインは、個別にのみ予測のプロセスをスピードアップしませんが、またクエリテンプレートのアラインメントの質はより信頼性の高い構造と機能の予測の結果、増加する。 タンパク質配列のドメイン境界は、NCBI CDD 24、PFAM 25またはInterProScanの26として自由に利用できる外部のオンラインプログラムを使用して予測することができます。また、アラインメントのスレッドLOMETS、クエリタンパク質のために使用可能な場合は、ドメインの境界を視覚的に（ステップ5.4を参照）上のスレッドのテンプレートに整列されていない残基の長いストレッチを識別することによって見つけることができます。これらのアライメントされていない領域は、主にドメインリンカー領域に対応しています。すべてのクエリーのドメインが整列してマルチドメインのテンプレートがテンプレートPDBライブラリですでに使用可能な場合は、クエリのタンパク質は、全長としてモデル化することができます。 外部制約を提供する<pクラスは、=&quot;jove_content&quot;&gt; I - TASSERの構造のアセンブリのシミュレーションは、主にテンプレートをスレッドLOMETSから収集された空間的な制約によって導かれる。テンプレートライブラリに優れたスレッドヒット（Norm. Z -スコア &gt; 1）を持っているクエリタンパク質については、派生空間的制約は、高精度であることが多い。とI - TASSERは、これらのタンパク質の高分解能構造モデルを生成します。逆に、弱い、またはまったくスレッディングヒット（Norm. Z -スコア &lt;1）を持つクエリのタンパク質で、収集された空間的な制約は、しばしばテンプレートとアライメントの不確実性のエラーが含まれている。これらのタンパク質のターゲットでは、ユーザーが指定した空間情報は、予測モデルの品質を向上させるために非常に役立ちます。ユーザーは、2つの方法でI - TASSERサーバーへの外部制約を提供することができます。 A.は、接触/距離制約を指定します。 NMRから、例えば実験的に特徴づけられる残基間コンタクト/距離、または架橋実験を、拘束のファイルをアップロードして指定することができます。例のファイルは図8に示すように、カラム1が拘束の種類を指定する場所、&quot;DIST&quot;または&quot;CONTACT&quot;すなわち。距離拘束（DIST）の場合は、列が2と4は、残基位置（i、j）を含む、列3と5は、残基と6列目の原子の種類を含む2つの指定された原子間の距離を指定します。接触拘束（CONTACT）の場合は、列2と3は接触にあるべき残基の位置を（i、j）を含んでいます。これらの連絡残基ペアの側鎖の中心間の距離は、PDBで知られている構造で観測された距離に基づいて決定されます。 I - TASSERは、構造精密化のシミュレーション中に指定された距離に近いこれらの原子のペアを描画しようとします。 B.は、タンパク質の構造のテンプレートを指定します。 LOMETSスレッドプログラムでは、クエリのprotのためのもっともらしい襞を見つけるために代表的なPDBライブラリを使用してくださいアイン。代表的な構造のライブラリを使用すると、配列構造のアラインメントを計算するために必要な時間を減らすために役立ちますが、それは良いテンプレートのタンパク質がライブラリ内に見落とされた場合、またはテンプレートが、それがであっても、LOMETSスレッドプログラムで識別されていない可能性がありますライブラリ内に存在する。これらのケースでは、ユーザーがテンプレートとして所望のタンパク質の構造を指定する必要があります。 追加のテンプレートとしてタンパク質の構造を指定するには、ユーザーがいずれかのPDBフォーマットされた構造のファイルをアップロードするか、PDB、ライブラリ内の堆積タンパク質の構造のPDB IDを指定することができます。 I - TASSERは召集プログラム23を使用してクエリテンプレートのアラインメントを生成し、指定されたユーザーテンプレートと構造の組立シミュレーションを導くためにLOMETSテンプレートの両方から空間的な制約を収集します。 LOMETS拘束具の精度が異なるターゲットが異なるため、LOMETSの拘束の重みは、容易に（相同）TAに強力です。体系的に私たちのベンチマークトレーニングにチューニングされているハード（非相同）ターゲット、のそれよりもrgets。 またユーザが独自のクエリテンプレートのアラインメントを指定することができます。 FASTA形式（図9A）及び3Dフォーマット（図9B）：サーバーには2つの形式で位置合わせを受け入れます。 FASTA形式は標準とで説明されています<a href="http://zhanglab.%20ccmb.med.umich.edu/FASTA/">http://zhanglab。 ccmb.med.umich.edu / FASTA /</a> 。 3Dフォーマットは、標準的なPDB形式（に似ています<a href="http://www.wwpdb.org/documentation/format32/sect9.html">http://www.wwpdb.org/documentation/format32/sect9.html</a> ）が、テンプレートから派生した2つの追加列はATOMの記録（図9B参照）に追加されます。 列1-30：アトム（C -αのみ）と残クエリシーケンスの名前。 列31から54：テンプレートの対応する原子からコピーされたクエリのC -α原子の座標。 列55から59：アライメントに基づいて、テンプレート内の対応する残基の数 列60から64：テンプレートの対応する残基名テンプレートの蛋白質を除外するタンパク質は、柔軟な分子であり、それらの生物学的活性を変更するには、複数の構造状態を採用することができます。例えば、多くのプロテインキナーゼと細胞膜タンパク質の構造は、 アクティブおよび非アクティブの両方コンフォメーションで解決されている。また、結合したリガンドの有無は、大きな構造的な動きを引き起こす可能性があります。テンプレートのすべての構造状態は、スレッド化プログラムのために似ているが、それは1つだけ特定の状態でテンプレートを使用してクエリをモデル化することが望ましい。サーバ上の新しいオプションは、ユーザが構造のモデリング時にテンプレートの蛋白質を除外することができます。また、この機能は、ユーザーがモデリングに使用するテンプレートの相同性レベルを選択できるようになります。ユーザーがテンプレートタンパク質frを除外することができます。OM I - TASSERライブラリ替え： A.は、配列同一性のカットオフを指定する ユーザーは、I - TASSERのテンプレートライブラリから相同蛋白質を除外するには、このオプションを使用することができます。相同性のレベルが配列同一のカットオフに基づいて設定されて、クエリとクエリ配列のシーケンスの長さで割ったテンプレートのタンパク質間の同一の残基の数、すなわち。提供する形で&quot;70％&quot;、配列同一性を持っているすべてのテンプレートのタンパク質&gt; 70％のユーザーがクエリタンパク質にたとえば、私は、予定I - TASSERのテンプレートライブラリから除外される。 B.特定のテンプレートの蛋白質を除外 特定のテンプレートのタンパク質が除外される構造のPDB IDを含むリストをアップロードすることによってI - TASSERのテンプレートライブラリから除外することができます。サンプルファイルは、図10に示します。同じタンパク質としてI - TASSER SE、PDBライブラリに複数のエントリとして存在することができますrverはデフォルトで指定されたテンプレート（列1の）だけでなく、アイデンティティーを持つライブラリから他のすべてのテンプレート&gt;指定したテンプレート〜90％が除外されます。また、ユーザーは、アイデンティティーを持つすべてのテンプレート&gt; 70％、指定されたテンプレートのタンパク質には除外されるなどの70％を、別のアイデンティティのカットオフを指定することができます。

<ol><li> FASTAは、モデル化されるタンパク質のアミノ酸配列（参照、フォーマット<a href="http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtml">http://www.ncbi.nlm.nih.gov/BLAST/fasta.shtmlを</a> ）。 </li><li>インターネットへのアクセスや、Webブラウザでパーソナルコンピュータ。 </li><li>予測三次構造と機能部位を分析するための分子可視化ソフトウエア、たとえばRasMolはまたはpymo​​lの、。 </li></ol>

a protocol for computer-based protein structure and function prediction

ゲノムシーケンシングプロジェクトは、その生物学的役割についての理解を向上させるために、それらの構造と機能の知識を必要とするタンパク質の配列の何百万を、暗号化されている。実験方法は、これらの蛋白質のごく一部のために詳細な情報を提供することができますが、計算モデルは実験的に未知されているタンパク質分子の大部分のために必要です。 I - TASSERサーバは、タンパク質の構造と機能の高解像度モデリングのためのオンラインワークベンチです。タンパク質配列を考えると、I - TASSERサーバからの典型的な出力は、二次構造予測が含まれ、各残基の溶媒露出度、スレッドと構造アラインメントによって検出された相同テンプレートのタンパク質、最大5つのフルレングスの三次構造モデル、および構造ベースの予測酵素の分類のための機能のアノテーション、遺伝子オントロジーの用語と蛋白質 - リガンド結合部位。すべての予測は信頼性スコアでタグ付けされている予測は実験データを知らなくてもあるか正確に伝えます。エンドユーザーの特殊な要求を容易にするために、サーバはユーザが指定した残基間の距離を受け入れ、対話的にI - TASSERのモデリングを変更するマップを連絡するチャネルを提供し、それはまた、ユーザーがテンプレートとして任意のタンパク質を指定するには、または任意のテンプレートを除外することができます構造の組み立てシミュレーション中のタンパク質。構造情報は、I - TASSER予測の質を改善する目的で実験的証拠または生物学的洞察に基づいて、ユーザーによって収集することができます。サーバは、最近の社会全体のCASP実験でタンパク質の構造と機能の予測のための最高のプログラムとして評価した。現在ありません&gt;オンラインI - TASSERサーバを使用している100カ国以上から20,000登録の科学者が。

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A Protocol for Computer-Based Protein Structure and Function Prediction

I - TASSERのパイプラインを使用してタンパク質の構造と機能解析に基づくコンピューターのためのガイドラインが記述されています。クエリタンパク質配列から始まる、3Dモデルは、複数のスレッドアラインメントを使用して生成され、構造組立シミュレーションを反復している。機能的な推論は、その後、既知の構造と機能を持つ蛋白質の一致に基づいて描画されます。

コンピュータベースのタンパク質の構造と機能の予測のためのプロトコル

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 ビュー.  University of Michigan. I - TASSERのパイプラインを使用してタンパク質の構造と機能解析に基づくコンピューターのためのガイドラインが記述されています。クエリタンパク質配列から始まる、3Dモデルは、複数のスレッドアラインメントを使用して生成され、構造組立シミュレーションを反復している。機能的な推論は、その後、既知の構造と機能を持つ蛋白質の一致に基づい?...

ビデオ: 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.

速度論と構造機能解析のアフィニティ定量のための適切なタンパク質濃度の重要性

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

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.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

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.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

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.

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル<sup&gt; 15</sup&gt; Nの代謝標識、免疫沈降法、定量的質量分析法と親和性調節

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.

プラントの脈管構造の三次元構造と機能を可視化するために、高分解能CTを用いた

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.

ハイスループットSHAPEを使用したRNA二次構造予測

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.

組換えタンパク質ベイトを用いてファージディスプレイおよび親和性選択のためのプロトコル

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.

タンパク質相互作用を発見したHaloTag技術を用いたタンパク質機能の特性評価

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.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

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.

タンパク質機能を調べるための誘導LAP-タグ付けされた安定な細胞株、時空間局在とタンパク質相互作用ネットワーク

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