この作品は、ゴードンとベティムーア財団、およびUNCF /メルク科学イニシアチブによって資金を供給された。

1。スクリーニングプレートの準備<ol><li> 25グラムのLB増殖培地、15グラム寒天、および1Lの蒸留水に1.5グラムカルボキシメチルセルロース（CMC）基板、および滅菌にオートクレーブを追加。 </li><li>オートクレーブ後、培地が適切な抗生物質を冷却し、追加することができますし、100μMの最終濃度になるようにIPTG。 </li><li> 5大正方形ペトリ皿/バイオアッセイ用トレイ（240 × 240 × 20mm以上大きい）にそれぞれ200mLの培地を注ぐ。プレートが完全に乾いてから。 </li></ol> 2。エンドグルカナーゼのライブラリの生成と画面<ol><li>エンドグルカナーゼ触媒ドメイン（複数可）のライブラリを生成します。これは、多くの方法で達成することができます（例えば、部位特異的またはランダム突然変異誘発、組換え、など）と研究者によって決定されます。 </li><li>彼らはlacオペレーターとT7プロモーターの制御下にあるようにベクターにライブラリのクローンを作成し、ペリプラズムへのターゲティングのためのpelBシグナル配列を含有する。適切なベクターの例は、NovagenからpET22b（+）です。 </li><li> E.にエンドグルカナーゼのライブラリを変換する大腸菌菌株BL21（DE3）および単一のコロニーのためのプレート。 </li><li> 37℃で一晩変換をインキュベート℃に</li><li>滅菌つまようじを使用して、適切な抗生物質を補充した400μLのLB培地を含む96ウェルディープウェルプレートにクローンを接種する。 250 rpmで振盪しながら37℃で一晩インキュベートする。 </li><li> -80℃で長期保存のための10％の最終濃度℃に96ウェル一晩培養して無菌のグリセロールを追加これがマスタープレートになります。 </li><li> 96ピンスタンプを用いて、IPTGとCMCを含むスクリーニングプレート上にプレート一晩培養を複製する。最高一晩培養の5セットまでは、各スクリーニングプレート上にスタンプすることができます。 </li><li>各96ウェルプレートの識別と向きがわかるようにスクリーニングプレートにラベルを付けます。 </li><li>室温（17〜25 ° C）一晩または目に見えるコロニーが形成されるまでにスタンプライブラリをインキュベートする。 </li><li>コロニーのすべてのトレースが削除されるまで蒸留水で皿をすすぐ。 </li><li>洗浄したプレートの上に、コンゴーレッド（水中に0.5％のCRを）注ぐ。プレートをカバーするだけの十分なCRを追加してください。 </li><li>室温で15分間インキュベートする。 CRのインキュベーションのための15分を超えないようにしてください。より長いインキュベーション時間が少なく明瞭ハローになります。 </li><li> CRを捨て、1MのNaClの寛大な量を（より十分なプレートをカバーする）を追加。 </li><li>室温で15分間インキュベートする。 </li><li> CMC分解ハローを明らかにするためにNaClを捨て。ハローのよりよい解決のために、NaClまたは複数のNaClを洗浄、より長いインキュベーション時間が必要になることがあります。 </li><li>滅菌つまようじを使用して、さらなる特性評価のためのマスタープレートから活性酵素のクローンを選ぶ。 </li></ol> 3。代表的な結果： このハイスループットスクリーニングのための読み出しの例を図3に示されています。クローンは劣化ゾーンのサイズに基づいて非アクティブな、弱い活性、および非常にアクティブとして識別することができます。既知の活性の酵素が基準としてライブラリに含まれている場合に特に便利です。ここに示すように、活性酵素の同定は、堅牢で再現性です。しかし、スクリーニングプレートの適切なラベル付けが非常に重要であることに注意してください。研究者は、° Cさらなる特徴付けのために-80℃で保存されたマスタープレートからそれらを選択するために、離れてコロニーを洗浄した後、アクティブな亜種を識別できる必要があります。最後に、人工的な対天然の基質上での活動の間に事前の関係を評価することは難しいことです。したがって、研究者は、蛋白質の適応度を決定するために工業的に関連する材料のすべての陽性クローンをテストする必要があります。 <img src="/files/ftp_upload/2942/2942fig1.jpg" alt="図1"/> 図1。スクリーニング手順の概略図概略。細胞は、エンドグルカナーゼライブラリで形質転換し、シングルコロニーを選択的にメッキが施されています。クローンは、-80℃（1）ストレージ℃、エンドグルカナーゼの発現を誘導するためにCMCとIPTGを含むプレート上で（2）レプリカのメッキ用96ウェルディープウェルプレートで一晩ピックアップと成長。アクティブなクローンは、-80℃で保存されたマスタープレートから取得さ<img src="/files/ftp_upload/2942/2942fig2.jpg" alt="図2"/> 図2 E.における真菌触媒ドメインのクローニングと発現大腸菌 。 （A）エンドグルカナーゼの発現ベクター。遺伝子はT7プロモーターの制御下で、pET22b（+）（Novagen）にクローニングした。 （B）右パネル：エンドグルカナーゼを発現するBL21（DE3）コロニー。左のパネル：コンゴーレッドによる洗浄および開発後のCMC分解ハロー。 <img src="/files/ftp_upload/2942/2942fig3.jpg" alt="図3"/> 図3。CMC /コンゴーレッドの画面からサ ​​ンプルの結果。洗浄とコンゴレッドの開発後にスクリーニングプレートから撮影した画像。の上のすべてのコロニーcreeningプレートは、同じようなサイズのものであった。

<ol>
	<li>Atsumi, S., Hanai, T., Liao, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fermentative+pathways+for+synthesis+of+branched-chain+higher+alcohols+as+biofuels.">Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.</a> Nature. 451, 86-89 (2008).</li><li>Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+and+biofuels.">Cellulases and biofuels.</a> Curr Opin Biotechnol. 20, 295-299 (2009).</li><li>Qin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+endoglucanase+II+from+Trichoderma+reesei+to+improve+the+catalytic+efficiency+at+a+higher+pH+optimum.">Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.</a> J Biotechnol. 135, 190-195 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+endoglucanase+III+(Cel12A)+from+Trichoderma+reesei.">Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.</a> Appl Microbiol Biotechnol. 83, 649-657 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+thermostable+fungal+cellulases+created+by+structure-guided+recombination.">A family of thermostable fungal cellulases created by structure-guided recombination.</a> Proc Natl Acad Sci. 106, 5610-5615 (2009).</li><li>Heinzelman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCHEMA+recombination+of+a+fungal+cellulase+uncovers+a+single+mutation+that+contributes+markedly+to+stability.">SCHEMA recombination of a fungal cellulase uncovers a single mutation that contributes markedly to stability.</a> J Biol Chem. 284, 26229-26233 (2009).</li><li>Lantz, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrea+jecorina+CEL6A+protein+engineering.">Hypocrea jecorina CEL6A protein engineering.</a> Biotechnol Biofuels. 8, 3-20 (2010).</li><li>Mahadevan, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutagenesis+and+CBM+engineering+of+Cel5A+(Thermotoga+maritima).">Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).</a> FEMS Microbiol Lett. 287, 205-211 (2008).</li><li>Nakazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+catalytic+domains+of+Trichoderma+reesei+endoglucanase+I,+II,+and+III,+expressed+in+Escherichia+coli.">Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli.</a> Appl Microbiol Biotechnol. 81, 681-689 (2008).</li><li>Wang, H., Jones, R. 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利害の衝突は宣言されません。

ここで説明するプロトコルは、最小限の操作で、アクティブな酵素の迅速かつ高スループットの識別が可能になります。アクティビティ検知とはかなり敏感であり、質的に、細胞内のアクティビティの量を反映している。その使いやすさはE.の機能的発現によって制限、酵素ライブラリの広い範囲のためのこの方法が適しています大腸菌 。さらに、この種の活動のスクリーニン?...

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>製品番号</th></tr><tr><td> IPTG </td><td>シグマ</td><td> I1284 </td></tr><tr><td>コンゴ赤</td><td>シグマ</td><td> C6277 </td></tr><tr><td>カルボキシメチルセルロース</td><td>シグマ</td><td> 360384 </td></tr><tr><td> NaClの</td><td>シグマ</td><td> S1679 </td></tr><tr><td>バクト寒天</td><td> BDバイオサイエンス</td><td> 214030 </td></tr><tr><td>バイオアッセイプレート</td><td>サーモサイエンティフィック</td><td> 240845 </td></tr></tbody></table>

high throughput screening of fungal endoglucanase activity in escherichia coli

セルラーゼ酵素（エンドグルカナーゼ、セロビオヒドロラーゼ、およびβ-グルコシダーゼ）順番に燃料アルコール1に変換することができるコンポーネントの糖類、に加水分解セルロース。再生可能エネルギーを提供するために、セルロース系バイオマスの酵素加水分解の可能性は、経済的な燃料生産の2のセルラーゼを設計するための努力を強化している。特に興味深いのは、すでに食品や繊維製品の処理のために工業的に使用されている真菌セルラーゼ3-8、です。 変異セルラーゼのライブラリの間でアクティブなバリアントを識別することは、エンジニアリングプロセスにとって重要です。アクティブな変異体は、さらに改良された特性について試験及び/または付加変異導入を行うことができる。真菌セルラーゼの効率的なエンジニアリングは、ネイティブの生物の遺伝的なツールの欠如によっておよび異種宿主中で酵素を発現における困難によって妨げられている。最近、森川らは、E.で表現するための手法を開発大腸菌 H. jecorina 3,9、大量のセルラーゼを分泌する能力を有する重要な産業の真菌からのエンドグルカナーゼの触媒ドメイン。機能性E.大腸菌の発現はまた、Macrophomina phaseolina 10とPhanerochaeteクリソ 11月12日を含む他の真菌、からセルラーゼのために報告されています。 我々は、E.の真菌エンドグルカナーゼ活性のハイスループットスクリーニングのための方法を提示大腸菌 。 （ 図1）このメソッドは、固形培地上に生育している細胞によるカルボキシメチルセルロース（CMC）の酵素分解を視覚化する一般的な微生物色素コンゴレッド（CR）を使用しています。活性測定は、安価な試薬、最小限の操作を必要とし、コロニーのサイトでの劣化のゾーン（&quot;ハロー&quot;）として、明確な結果が得られます。酵素活性の定量的測定がこの方法によって決定することはできませんが、我々は、ハローのサ​​イズはセル内の総酵素活性と相関することを発見した。個々の陽性クローンのさらなる特徴は、相対的な蛋白質の適応度を決定します。 伝統的な細菌の全細胞CMC / CR活性アッセイ13はクロスコンタミネーション、または大規模な実験にはあまり適しているCMC寒天井戸、でインキュベート文化の対象となるコロニー、上に寒天を含むCMCを注ぐ伴います。ここでは、セルラーゼ活性のための14の既存の洗浄方法を変更する改良されたプロトコルを報告する：CMC寒天プレート上で増殖した細胞は、前のCRの染色に削除されます。我々のプロトコルが大幅にクロスコンタミネーションを低減し、クローンの数千の迅速なスクリーニングを可能にする、非常にスケーラブルです。 H.に加えてjecorina酵素は 、我々が表明していると、このプロトコルは、生物の範囲から酵素に適用可能であることを示唆し、Thermoascus aurantiacusとペニシリウムdecumbens（ 図2に示されている）からエンドグルカナーゼの変異体をスクリーニングした。

Watch this Scientific Journal Video about High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli at JoVE.com

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

私達は中の真菌エンドグルカナーゼ活性をスクリーニングするために、低コスト、高スループットの方法を説明します<em&gt; E.大腸菌。</em&gt;メソッドは、基板の劣化のシンプルなビジュアル読み出しに依存している酵素の精製を必要とせず、非常にスケーラブルです。これは、酵素変異体の大規模なライブラリーの迅速なスクリーニングが可能になります。

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

high-throughput-screening-fungal-endoglucanase-activity-escherichia

Research

JoVE Journal

Biology

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

20.4K ビュー.  California Institute of Technology. 私達は中の真菌エンドグルカナーゼ活性をスクリーニングするために、低コスト、高スループットの方法を説明します E.大腸菌。メソッドは、基板の劣化のシンプルなビジュアル読み出しに依存している酵素の精製を必要とせず、非常にスケーラブルです。これは、酵素変異体の大規模なライブラリーの迅速なスクリーニングが可能になりま?...

ビデオ: High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

生物学

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

メタゲノムライブラリからセルラーゼ活性をBiominingためのハイスループットスクリーニング

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向き整流カリウムチャネルの小分子モジュレーターのためのハイスループットスクリーニング

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

で産生された組換えジスルフィド豊富な毒タンパク質に印加される高スループット定量的発現スクリーニングおよび精製<em&gt; E.大腸菌の</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

タンパク質の共発現の多面的メリットで<em&gt;エシェリヒア·コリ</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...