Name: a yeast 2-hybrid screen in batch to compare protein interactions
Uploaded: 2018-06-06T14:30:00.000+00:00
Duration: 14 min 23 s
Description: Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

NGS ライブラリの準備およびシーケンスのための人間遺伝学研究所内スタッフに感謝いたします。ここで作られた Y2H プラスミド ライブラリのゲノム ライブラリー フラグメントの準備で彼女の専門知識を Einat snir/を感謝いたします。この仕事は健康の国民の協会によって支えられた: NIH R21 EB021870 01A1、NSF 研究プロジェクト助成: 1517110。

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrY2H-seq:+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping.">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li><li>Estojak, J., Brent, R., Golemis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+two-hybrid+affinity+data+with+in+vitro+measurements.">Correlation of two-hybrid affinity data with in vitro measurements.</a> Molecular and Cellular Biology. 15 (10), 5820-5829 (1995).</li><li>Rajagopala, S. V., Hughes, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-Polyacrylamide+Gel+Electrophoresis+of+Proteins.">SDS-Polyacrylamide Gel Electrophoresis of Proteins.</a> Cold Spring Harbor Protocols. 2006 (4), (2006).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the National Academy of Sciences USA. 76 (9), 4350-4354 (1979).</li><li>Alegria-Schaffer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting+using+chemiluminescent+substrates.">Western blotting using chemiluminescent substrates.</a> Methods in Enzymology. 541, 251-259 (2014).</li><li>Lee, P. 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著者がある何も開示するには

ここで最適化されたメソッドを使用してバッチで Y2H アッセイを実行する方法のためのガイドを提供します。開始ライブラリの代表を選択下に置かれると酵母の人口には変動を制限するための選択を受けること開始の酵母集団の十分なされているかを確認する手順をいくつかの重要なステップがあります。.重要なは、これらのベンチマークは、比較的適応方法やこのアプローチになり、ほと?...

細胞生物学的プロセスの完全な理解は、その分子機構の根底にある蛋白質相互作用ネットワークの検索に依存します。蛋白質の相互作用を識別する 1 つの方法は興味の蛋白質の 2 つの領域は互いにバインド1一度機能しているキメラ転写因子を組み立てることによって動作する酵母 2 ハイブリッド (Y2H) 試金。Y2H の典型的なスクリーンがプラスミド転写活性化因子に融合相互作用の蛋白質の両方図書館を収容する酵母の人口を作成することによって実行されます (例えば.、'捕食' 融合タンパク質) とタンパク質から成る特定の '餌' プラスミド興味の DNA 結合ドメイン (例えば、Gal4 上流活性化シーケンスにバインド Gal4 DNA 結合ドメイン) に溶けます。Y2H アプローチの主な利点の 1 つは、比較的簡単だし、ルーチンワーク分子生物2の典型的な研究室で実施する安価な装備です。しかし、伝統的に実行されると、ユーザーは正の Y2H 相互作用の選択により発生する個々 のコロニーをサンプリングします。これは、調査することができますライブラリ '捕食' クローンの数を厳しく制限されます。この問題は、特定の相互作用する獲物の豊富さが非常に高い他、低豊富な獲物プラスミドからの相互作用を検出する機会を減少と関連した合成されます。
プロテオームの包括的なカバレッジの Y2H 原理を使用して 1 つのソリューションは、知られている個々 の獲物プラスミドを含む配列を前記デジタルに尋問できるマトリックス形式のアプローチの使用です。ただし、このようなアプローチは蛋白質またはドメイン3の少数のインタラクトームを定義する際に興味を持っている個々 の捜査官に容易にアクセスや費用対効果のないインフラストラクチャが必要です。さらに、複数のフラグメントの相互作用の蛋白質を符号化するかもしれない非常に複雑な獲物ライブラリは、非現実的なサイズにこのような行列の配列のサイズを拡大します。代わりに、バッチで複雑なライブラリとアッセイを行い、大規模な並列高スループット シーケンス4を使用して相互作用するクローンの存在を評価します。これはアッセイの典型的なを使用して複数のコロニーで発生する獲物プラスミドの存在に適用できる Y2H 形式アプローチする酵母の細胞融合蛋白質の相互作用のペアを住宅プレート5,6に成長すること。この一般的な考えは、両方の複数の餌のクエリを増やすし、同じ時間7,8のコンポーネントを獲物に強調することができます。
それでも、多くの調査は簡単にまだ努力に注力ほんのタンパク質 '餌' を必要とし、単一の複雑な獲物ライブラリの包括的な半定量的なクエリによって恩恵を受けることができます。我々 は開発し、バッチ形式4で Y2H 原理を用いた大規模タンパク質相互作用研究を実行するアプローチを検証します。Y2H 相互作用9の相対的な強さのためのプロキシとして特定の獲物プラスミドの膨張率を使用します。集団内のすべてのプラスミドのディープ シーケンスが正常な成長を受けるまたは選択成長条件は、強くて弱い Y2H 相互作用を得られるクローンの完全なマップを生成します。インターアクターのレパートリーを入手し、直接複数の餌プラスミド間で比較することができます。Deepn 深さ (評価のタンパク質ネットワークの動的な濃縮) と呼ばれる作成されたワークフローは、対別のもの 1 つのタンパク質の比較を許可する蛋白質を識別する同じ獲物ライブラリから差分 interactomes を識別するためにこのように使用できます。
Deepn 深さを示すここでは、図 1に記載されている、その使用を容易にする実験室方法の改善を紹介。大幅な改善が含まれます。
獲物酵母集団の生成します。プラスミド獲物ライブラリの分布が同じ別の餌プラスミドと酵母の集団の生成 deepn 深さの重要な要件の 1 つです。獲物プラスミド ライブラリの同等基準集団が異なる餌の interactomes 間の正確な比較を行うために不可欠です。これは最高のライブラリ プラスミドは既に建物半数体酵母の人口で、その人口に与えられた餌のプラスミドの移動は、二倍体を生成する交配によって達成されるとき達成します。ここでは、我々 は半数体酵母の市販のライブラリを使用してこのような集団を作る方法で、明確な指針を提供します。我々 は二倍体数が多いを生成するメソッドを発見したが、これら商用ライブラリを含む酵母菌の全体的な交尾効率が低かった。したがって、交配反応あたりはるかに多くの切片が得られます獲物ライブラリを収容できる新しいひずみを構築しました。
新しい餌プラスミドの設定をします。エクスプレス '餌' 融合蛋白質で構成される興味の DNA 結合ドメインとタンパク質の多くの現在のプラスミドは、2 μ ベース、コピー数を増幅させることです。このコピーの数は、人口の非常に変数をでき、Y2H 転写応答の可変性に 。これは、ターンでは選択範囲の下のセルの成長の応答に基づいて特定の蛋白質の相互作用の強さを測定する能力が偏る可能性があります。これは、部分的アドレスのいくつかは以前市販 pDEST3210など記載されている低コピー プラスミドを用いた。上流の餌フラグメントの両方のクローンを作成ことができます Kanr 耐性遺伝子を運ぶTRP1セントロメア ベース低コピー プラスミド内 Gal4 DNA 結合ドメイン融合蛋白質を作り出す新しい餌プラスミド (pTEF GBD) を構築したとGal4 DNA 結合ドメインの下流。
新しい高密度 Y2H フラグメント ライブラリ。家 Y2H 獲物ライブラリに新しいプラスミドを構築し、ゲノム DNA の断片をランダムにせん断の酵母から作られた非常に複雑な Y2H ライブラリをビルドするために使用します。シーケンス解析は、このライブラリが前述の酵母ゲノム Y2H プラスミド ライブラリ11よりも遠く複雑以上 100 万の異なる要素を持っていたことを示した。この新しいライブラリ DEEPN ワークフローが堅牢な信頼性が高く、再現性のある方法で多くの異なるプラスミドを持つ複雑なライブラリに対応することを示すことができました。

<table><tbody><tr><td>Illumina HiSeq 4000</td><td>Illumina</td><td></td><td>deep sequencing platform</td></tr><tr><td>Monoclonal anti-HA antibodies</td><td>Biolegend</td><td>901514</td><td>Primary Antibody to detect expression of HA in pGal4AD constructs</td></tr><tr><td>Polyclonal anti-myc antibodies</td><td>QED Biosciences Inc</td><td>18826</td><td>Primary Antibody to detect expression of MYC in pTEF-GBD constructs</td></tr><tr><td>NarI</td><td>New England BioLabs</td><td>R0191S</td><td></td></tr><tr><td>EcoRI-HF</td><td>New England BioLabs</td><td>R3101S</td><td></td></tr><tr><td>BamHI-HF</td><td>New England BioLabs</td><td>R3236S</td><td></td></tr><tr><td>XhoI</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>Polyethylene Glycol 3350, powder</td><td>J.T. Baker</td><td>U2211-08</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Trevigen, Inc sold by Fisher Scientific</td><td>50-948-286</td><td>carrier DNA for yeast transformation section 3.2.1.</td></tr><tr><td>Kanamycin Monosulfate</td><td>Research Products International</td><td>K22000</td><td></td></tr><tr><td>LE Agarose</td><td>GeneMate</td><td>E-3120-500</td><td>used for making DNA agarose gels</td></tr><tr><td>Sodium Chloride</td><td>Research Products International</td><td>S23025</td><td></td></tr><tr><td>Tryptone</td><td>Research Products International</td><td>T60060</td><td></td></tr><tr><td>D-Sorbitol</td><td>Research Products International</td><td>S23080</td><td></td></tr><tr><td>Lithium Acetate Dihydrate</td><td>MP Biomedicals</td><td>155256</td><td></td></tr><tr><td>Calcium Chloride</td><td>ThermoFisher</td><td>C79</td><td></td></tr><tr><td>EDTA Sodium Salt</td><td>Research Products International</td><td>E57020</td><td></td></tr><tr><td>Yeast Extract Powder</td><td>Research Products International</td><td>Y20020</td><td></td></tr><tr><td>Yeast Nitrogen Base (ammonium sulfate) w/o amino acids</td><td>Research Products International</td><td>Y20040</td><td></td></tr><tr><td>CSM-Trp-Leu+40ADE</td><td>Formedium</td><td>DCS0789</td><td></td></tr><tr><td>CSM-Trp-Leu-His+40ADE</td><td>Formedium</td><td>DCS1169</td><td></td></tr><tr><td>CSM-Leu-Met</td><td>Formedium</td><td>DCS0549</td><td></td></tr><tr><td>CSM-Trp-Met</td><td>Bio 101, Inc</td><td>4520-922</td><td></td></tr><tr><td>L-Methionine</td><td>Formedium</td><td>DOC0168</td><td></td></tr><tr><td>Adenine</td><td>Research Products International</td><td>A11500</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Research Products International</td><td>G32045</td><td></td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td>used for making media plates in section 1</td></tr><tr><td>Peptone</td><td>Research Products International</td><td>P20240</td><td></td></tr><tr><td>3-amino-1,2,4 Triazole</td><td>Sigma</td><td>A8056</td><td></td></tr><tr><td>2-Mercaptoehanol (BME)</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>Zymolyase 100T</td><td>USBiological</td><td>Z1004</td><td></td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma</td><td>P8281</td><td></td></tr><tr><td>Phenol:Chloroform:IAA</td><td>Ambion</td><td>AM9732</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>238074</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories, INC</td><td>2716</td><td></td></tr><tr><td>RNAse A</td><td>ThermoFisher</td><td>EN0531</td><td></td></tr><tr><td>Urea</td><td>Research Products International</td><td>U20200</td><td></td></tr><tr><td>SDS</td><td>Research Products International</td><td>L22010</td><td></td></tr><tr><td>glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Tris-HCl</td><td>Gibco</td><td>15506-017</td><td></td></tr><tr><td>bromophenol blue</td><td>Amresco</td><td>449</td><td></td></tr><tr><td>Gibson Assembly Cloning Kit</td><td>New England Biolabs</td><td>E5510S</td><td>Rapid assembly method for cloning of plasmids in section 2</td></tr><tr><td>NEBNext High-Fidelity 2x PCR Master Mix</td><td>New England Biolabs</td><td>M0541S</td><td>Used for amplification of products for Gibson Assembly in Section 2.3 as well assample preparation for DEEPN deep sequencing in section 6.2.1</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>0492-5G</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td>Used for purification of pcr products in section 6.2.3</td></tr><tr><td>Qiaquick DNA Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Used for purification of digested pTEF-GBD in section 2.1</td></tr><tr><td>KAPA Hyper Prep kit</td><td>KAPA Biosystems</td><td>KK8500</td><td>preparation kit for deep sequencing</td></tr><tr><td>Codon optimization</td><td>http://www.jcat.de</td><td></td><td></td></tr><tr><td>Codon optimization</td><td>https://www.idtdna.com/CodonOpt</td><td></td><td></td></tr><tr><td>gBlocks</td><td>Integrated DNA Technologies</td><td></td><td>DNA fragments used for cloning in Section 2.2</td></tr><tr><td>Strings</td><td>Thermofisher</td><td></td><td>DNA fragments used for cloning in Section 2.2</td></tr><tr><td>GenCatch Plasmid DNA mini-prep Kit</td><td>EPOCH Life Sciences</td><td></td><td>Used to prepare quantities of DNA in Section 2.3</td></tr><tr><td>Covaris E220</td><td>Covaris</td><td></td><td>high performance ultra-sonicator in section 7</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;- CGGTCTT&lt;br/&gt; CAATTTCTCAAGTTTCAG -3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 5&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-GAGTAACG&lt;br/&gt; ACATTCCCAGTTGTTC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 5&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-CACCGTAT&lt;br/&gt; TTCTGCCACCTCTTCC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 3&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligo nucelotide 5&amp;rsquo;-GCAACCGC&lt;br/&gt; ACTATTTGGAGCGCTG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>used for pcr amplification and sequencing 3&#039; insert pTEF-GBD during plasmid construction</td></tr><tr><td>oligonucleotide 5&amp;rsquo;-GTTCCGATG&lt;br/&gt; CCTCTGCGAGTG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>5&#039; Pimer used for insert amplification of pGAL4AD</td></tr><tr><td>oligonucelotide 5&amp;rsquo;-GCACATGCT&lt;br/&gt; AGCGTCAAATACC-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>3&#039; Pimer used for insert amplification of pGAL4AD</td></tr><tr><td>oligonucelotide 5&amp;rsquo;-ACCCAAGCA&lt;br/&gt; GTGGTATCAACG-3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>5&#039; Pimer used for insert amplification of pGADT7</td></tr><tr><td>oligonucelotide 5&amp;rsquo;- TATTTAGA&lt;br/&gt; AGTGTCAACAACGTA -3&amp;rsquo;</td><td>Integrated DNA Technologies or Thermofisher</td><td></td><td>3&#039; Pimer used for insert amplification of pGADT7</td></tr><tr><td>PJ69-4A MatA yeast strain</td><td>http://depts.washington.edu/yeastrc/ James P, Halladay J, Craig EA: Genomic Libraries and a host strain designed for highly efficient two-hybrid selection in yeast. GENETICS 1996 144:1425-1436</td><td></td><td>MATA leu2-3,112 ura3-52 trp1-901 his3-200 gal4D, gal80D, GAL-ADE2 lys2::GAL1-HIS3 met2::GAL7</td></tr><tr><td>pTEF-GBD</td><td>Dr. Robert Piper Lab</td><td></td><td>Gal4-DNA binding doimain expression plasmid</td></tr><tr><td>pGal4AD (pPL6343)</td><td>Dr. Robert Piper Lab</td><td></td><td>Gal4-activation domain expression plasmid</td></tr><tr><td>100 mm petri dishes</td><td>Kord-Vallmark sold by VWR</td><td>2900</td><td></td></tr><tr><td>125 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63270</td><td></td></tr><tr><td>250 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63271</td><td></td></tr><tr><td>1,000 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63274</td><td></td></tr><tr><td>2,000 mL PYREX Erlenmeyer flask</td><td>Fisher Scientific</td><td>S63275</td><td></td></tr><tr><td>20 X 150 mm Disposable Culture Tube</td><td>Thermofisher</td><td>14-961-33</td><td></td></tr><tr><td>pipet-aid</td><td>Drummond</td><td>4-000-100</td><td></td></tr><tr><td>5 mL Serological Pipette</td><td>Denville</td><td>P7127</td><td></td></tr><tr><td>10 mL Serological Pipette</td><td>Denville</td><td>P7128</td><td></td></tr><tr><td>25 mL Serological Pipette</td><td>Denville</td><td>P7129</td><td></td></tr><tr><td>1,000 mL PYREX Griffin Beaker</td><td>Fisher Scientific</td><td>02-540P</td><td></td></tr><tr><td>1,000 mL PYREX Reuasable Media Storage Bottle</td><td>Fisher Scientific</td><td>06-414-1D</td><td></td></tr><tr><td>1,000 mL graduated cylinder</td><td>Fisher Scientific</td><td>08-572-6G</td><td></td></tr><tr><td>SpectraMax 190</td><td>Molecular Devices</td><td></td><td>used to measure the Optical Density of cells</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td>ND-2000</td><td>Spectrophotometer used to quantify amount of DNA</td></tr><tr><td>Electronic UV transilluminator</td><td>Ultra Lum</td><td>MEB 20</td><td>used to visualize DNA in an Ethidium Bromide agarose gel</td></tr><tr><td>P1000 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123602G</td><td></td></tr><tr><td>P200 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123601G</td><td></td></tr><tr><td>P20 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F123600G</td><td></td></tr><tr><td>P10 Gilson PIPETMAN</td><td>Fisher Scientific</td><td>F144802G</td><td></td></tr><tr><td>1250 &amp;micro;L Low Retention Pipette Tips</td><td>GeneMate</td><td>P-1236-1250</td><td></td></tr><tr><td>200 &amp;micro;LLow Retention Pipette Tips</td><td>VWR</td><td>10017-044</td><td></td></tr><tr><td>10 &amp;micro;L XL Low Retention Pipette Tips</td><td>VWR</td><td>10017-042</td><td></td></tr><tr><td>50 mL conical tube</td><td>VWR</td><td>490001-627</td><td></td></tr><tr><td>15 mL conical tube</td><td>VWR</td><td>490001-621</td><td></td></tr><tr><td>cell scraper</td><td>Denville Scientific</td><td>TC9310</td><td></td></tr><tr><td>1.5 mL Microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>HCl</td><td>Fluka Analytical</td><td>318949-1L</td><td></td></tr><tr><td>NaOH</td><td>J.T. Baker</td><td>5674-02</td><td></td></tr><tr><td>Wooden applicators</td><td>Solon Care</td><td>55900</td><td></td></tr><tr><td>Eppendorf microcentrifuge 5424</td><td>Fisher Scientific</td><td>05-400-005</td><td>microcentrifuge</td></tr><tr><td>Sorvall ST16R</td><td>Thermo Fisher Scientific</td><td>75004381</td><td>benchtop centrifuge</td></tr><tr><td>Amersham ECL Rabbit IgG, HRP-linked whole Ab (from donkey)</td><td>GE Healthcare</td><td>NA934-1ML</td><td>Secondary Antibody</td></tr><tr><td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td><td>GE Healthcare</td><td>NA931-1ML</td><td>Secondary Antibody</td></tr><tr><td>SuperSignal West Pico Chemiluminescent Substrate</td><td>Thermo Fisher Scientific</td><td>34080</td><td>ECL detection solution</td></tr><tr><td>Isotemp Incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Incubator</td></tr><tr><td>Mutitron 2</td><td>INFORS HT</td><td></td><td>Shaking incubator</td></tr><tr><td>Isotemp Digital-Control Water Bath Model 205</td><td>Fisher Scientific</td><td></td><td>water bath</td></tr><tr><td>Y2H mouse cDNA library in Y187 (pan tissue)</td><td>Clontech</td><td>630482</td><td>commercially available cDNA Library</td></tr><tr><td>Y2H mouse cDNA library in Y187 (mouse brain)</td><td>Clontech</td><td>630488</td><td>commercially available cDNA Library</td></tr><tr><td>pGADT7 AD Vector</td><td>Clontech</td><td>630442</td><td>commercially available AD Vector housing many cDNA libraries</td></tr><tr><td>pGBKT7 DNA-BD Vector</td><td>Clontech</td><td>630443</td><td>commercially available DNA-BD Vector</td></tr><tr><td>Biolase DNA Polymerase</td><td>Bioline</td><td>BIO-21042</td><td>DNA polymerase used for section 2.4</td></tr><tr><td>GeneMate GCL-60 Thermal Cycler</td><td>BioExpress</td><td>P-6050-60</td><td>pcr machine</td></tr><tr><td>TempAssure 0.5 mL PCR tubes</td><td>USA Scientific</td><td>1405-8100</td><td></td></tr></tbody></table>

a yeast 2-hybrid screen in batch to compare protein interactions

酵母 2 ハイブリッド法によるタンパク質-タンパク質相互作用のためのスクリーニングは、効果的なツールを長年が、その使用は主として相互作用する候補者のライブラリで濃縮され高い高親和性インターアクターの発見に制限されています。伝統的な形式の酵母 2 ハイブリッド試金は低親和性インターアクターを発見可能性があります低金詰りで実施を分析するあまりにも多くの植民地を得ることができます。また、別の餌プラスミドに対して同じライブラリの包括的かつ完全な尋問、なし比較分析を実現できません。これらの問題のいくつかは、アレイ獲物ライブラリを使用して対処できる、コストとインフラストラクチャは、このような画面を操作するために必要なことが禁止できます。代わりに、我々 は同時に過渡の多数を明らかにする酵母 2 ハイブリッド試金を適応しているし、戦略を利用した単一の画面内で静的な蛋白質の相互作用に deepn 深さ (評価のタンパク質ネットワークの動的な濃縮) と呼ばれています。ハイスループット DNA の配列と相互作用のパートナーをエンコード プラスミドの人口の進化に従ってくださいに計算が組み込まれています。ここでは、カスタマイズされた試薬と簡単にかつ低コストで実行する deepn 深さが画面を可能にするプロトコルについて述べる。

Y2H 試金は蛋白質: 蛋白質の相互作用を見つけるため広く使用されているし、いくつかの適応やシステムが開発されています。ほとんどの部分については、これらの従来のアプローチを成功させるに役立つ同じ考慮事項が deepn 深さが重要です。重要なベンチマークのいくつかが含まれます: DNA 結合ドメインの発現融合蛋白質、スプリアスの低バック グラウンド彼 + 空?...

Watch this Scientific Journal Video about A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions at JoVE.com

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

蛋白質の相互作用を比較するバッチの酵母 2 ハイブリッド スクリーン

酵母 2 ハイブリッド スクリーンのバッチ処理は、獲物の融合蛋白質の非常に複雑なセットを持つ複数の餌の蛋白質の相互作用のプロファイルの直接比較のためことができます。ここでは、我々 は洗練された方法、新しい試薬とこのような画面の使用を実装する方法について説明します。

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

a-yeast-2-hybrid-screen-in-batch-to-compare-protein-interactions

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

13.3K ビュー.  University of Iowa. 酵母 2 ハイブリッド スクリーンのバッチ処理は、獲物の融合蛋白質の非常に複雑なセットを持つ複数の餌の蛋白質の相互作用のプロファイルの直接比較のためことができます。ここでは、我々 は洗練された方法、新しい試薬とこのような画面の使用を実装する方法について説明します。

ビデオ: A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

異種タンパク質複合体-DNA相互作用研究のための改変酵母1ハイブリッドシステム

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

生化学

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

バッチ酵母 2 ハイブリッド スクリーンからシーケンス データの情報解析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

遺伝学

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

免疫・感染学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

生物学

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

以下のための遺伝学的および生化学的アプローチ<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;タンパク質のオリゴマーの評価：リアノジン受容体のケーススタディ

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

酵母ツーハイブリッド法による生体分子間相互作用研究 

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

著者スポットライト:酵母におけるInteraction-null変異体の分離の強化

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

蛋白質の相互作用を比較するバッチの酵母 2 ハイブリッドスクリーン

要約

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この動画の章

異種タンパク質複合体-DNA相互作用研究のための改変酵母1ハイブリッドシステム

バッチ酵母 2 ハイブリッドスクリーンからシーケンスデータの情報解析

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

以下のための遺伝学的および生化学的アプローチ

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

蛋白質の相互作用を比較するバッチの酵母 2 ハイブリッド スクリーン

要約

さらに動画を探す

この動画の章

異種タンパク質複合体-DNA相互作用研究のための改変酵母1ハイブリッドシステム

バッチ酵母 2 ハイブリッド スクリーンからシーケンス データの情報解析

ホスト病原体のタンパク質 - タンパク質相互作用の風景を特徴づけるために比較的アプローチ

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

以下のための遺伝学的および生化学的アプローチ

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

Yeast Two-Hybrid Assayを用いたInteraction-Null/Impaired Mutantsの単離

蛋白質の相互作用を比較するバッチの酵母 2 ハイブリッドスクリーン

バッチ酵母 2 ハイブリッドスクリーンからシーケンスデータの情報解析