この研究は、ポーランドのクラクフにある国立科学センター(NCN)からの助成金番号2016/23/D/NZ3/01314によって支援されました。

発現プラスミドの作製
<ol><li>トポロジー予測ツールを使用して、目的のタンパク質の膜トポロジーを調べます。</li>
	<li>融合タンパク質がゴルジ膜に挿入されると、大きい断片と小さい断片が細胞質に面するようにクローニング戦略を設計します。ここで提示する場合のように、目的のタンパク質のN末端とC末端の両方が細胞質的に配向している場合は、8つの可能な方法でタンパク質にタグを付けます( 図1Bを参照)。目的のタンパク質の一方または両方のN末端またはC末端が光度配向している場合は、タグ付けから除外します。</li>
	<li>標準的なクローニングプロトコールに従って、目的の遺伝子を適切な発現ベクター( 材料表を参照)にサブクローニングします。 メモ: 近接性の欠如、最適でない方向、または空間的制約のために、一部のタグ付けオプションが機能しない可能性があるため、考えられるすべての向きをテストすることをお勧めします。</li>
</ol>2. 発現プラスミドの細胞への一過性トランスフェクション
<ol><li>トリプシン処理によって付着したHEK293T細胞培養物を回収し、専用の完全増殖培地に細胞を再懸濁する。細胞(2 x 104/100 μL/ウェル)を透明な底の白い側面96ウェルプレートにプレートします。ウェルの総数を調整して、反復を含むすべての試験済みの組み合わせと対照に対応します。 メモ: プレートの内側の 60 ウェルのみを使用して、熱シフトを最小限に抑え、一晩の蒸発を回避してください。 材料表に示されているようなポリD-リジンコーティングプレートを使用することを強く推奨し、そうしないとその後の洗浄ステップ中に細胞が剥離する可能性がある。</li>
	<li>細胞を標準条件(37°C、5%CO2)で一晩培養する。</li>
	<li>翌日、ポイント1.1で得られた発現プラスミドの所望の組み合わせで細胞をトランスフェクトする。<ol>
<li>発現プラスミドを無血清培地( 材料表を参照)で希釈し、各構築物について6.25 ng/μLに希釈する。</li>
		<li>脂質ベースのトランスフェクション試薬を適切な脂質対DNA比で添加し、メーカーの指示に従ってインキュベートします。</li>
		<li>8 μL の脂質:DNA混合物を指定されたウェルに加える。プレートの内容物を緩やかな回転で混ぜる。これにより、両方の発現構築物を50ng/ウェルでトランスフェクションする。</li>
	</ol>
</li>
	<li>細胞を標準条件(37°C、5%CO2)で20〜24時間培養する。 注:細胞をより長い時間培養すると、融合タンパク質発現のレベルが高くなり、断片間の非特異的な会合が促進される可能性があります。</li>
</ol>3. 媒体交換
<ol><li>翌日、馴化培地を各ウェル内の無血清培地100 μLと交換する。培地交換時に細胞が剥離していないことを確認してください。 注:このステップは、フリマジン作動溶液を添加する2〜3時間前に行う必要があります。血清離脱は、フリマジンの自律発光によって引き起こされるバックグラウンドを最小限に抑える。</li>
</ol>4.フリマジン作業溶液の調製
<ol><li>測定の直前に、1容量のフリマジンを19容量の希釈バッファー(20倍希釈)と混合する。 注:調製されるフリマジン作動溶液の総量は、分析される個々のウェルの数に依存する(フリマジン作動溶液は1:5の比率で細胞培養培地に添加されるので、100μLの無血清培地で以前に充填された各ウェルに25μLのフリマジン作動溶液を添加するべきである)。</li>
	<li>フリマジン作業溶液を指定されたウェル(25 μL/ウェル)に加えます。プレートを手で、またはオービタルシェーカー(例えば、300〜500rpmで15秒)を使用して静かに混合する。</li>
</ol>5. 発光の測定
<ol><li>プレートをルミネッセンスマイクロプレートリーダーに挿入します。<ol>
<li>37°Cで行われる実験については、示された温度でプレートを10〜15分間平衡化する。</li>
	</ol>
</li>
	<li>分析する井戸を選択します。</li>
	<li>積分時間0.3秒で発光を読み取る。必要に応じて、発光を最大2時間監視し続けます。</li>
</ol>6. データ解析
<ol><li>試験済みと対照のすべての組み合わせの平均値と標準偏差を計算します。</li>
	<li>多重比較を伴う一元配置分散分析を使用してデータを分析します。</li>
	<li>目的の組合せについて得られた平均発光を、対応する陰性対照について得られた平均発光で割ることによって、フォールド変化値を計算する。結果を評価します。 注:ここで提案するデータ分析へのアプローチは、試験された組み合わせについて得られた発光が、対応する対照の組み合わせについて得られた発光よりも統計的に有意に高く、同時に、これら2つの値の比が10を超える場合に、相互作用を主張できると仮定する。</li>
</ol>

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著者らは開示するものは何もありません。

ここでは、分割ルシフェラーゼ相補アッセイを使用して、NSTなどのゴルジ常駐III型膜タンパク質間に形成される異種複合体の実証を可能にする詳細なプロトコルを提供します。データ解析および解釈への提案されたアプローチは、目的のタンパク質の組み合わせについて得られた発光を、より大きな断片と融合した目的のタンパク質の1つおよびより小さい断片と融合した細菌起源の制御タン...

この原稿では、スプリットルシフェラーゼ相補アッセイの最新のバリアントを使用して、一過性にトランスフェクトされたヒト細胞におけるゴルジ体常駐III型膜タンパク質間の異種相互作用の存在をチェックするための段階的なプロトコルについて説明しています。この手順は、ヌクレオチド糖トランスポーター(NST)に対して最も広範囲に試験されていますが、N末端および/またはC末端が細胞質に面している他のゴルジ常駐III型膜タンパク質についても肯定的な結果を得ることができました。
我々の研究グループは、巨大分子のグリコシル化におけるNSTの役割を探究している。NSTはゴルジ体および/またはER常駐III型膜タンパク質であり、N末端およびC末端はオルガネラー膜の細胞質側を向いています1。NSTは、糖転移酵素に基質を供給するために、オルガネラ膜を横切ってヌクレオチド活性化糖を運ぶと考えられている。NSTは二量体および/またはそれ以上のオリゴマー2、3、4、5、6、7、8、9、10を形成する。さらに、異なるNST間の異種相互作用も報告されている6,11。NSTはまた、機能的に関連するグリコシル化酵素との複合体を形成することが実証された12、13、14。私たちは、NSTと機能的に関連するゴルジ体常駐タンパク質の相互作用を研究するために、現在使用されている蛍光寿命イメージング(FLIM)ベースのFRETアプローチに代わるものを探し、スプリットルシフェラーゼ相補アッセイをテストすることに決めました。これにより、NSTと機能的に関連するグリコシル化酵素との間の新しい相互作用を同定することができました9。
スプリットルシフェラーゼ相補アッセイの最新の改変であるNanoBiTは、ここで提示されたプロトコルで使用されています15。これは、2つの断片からのルシフェラーゼ酵素(例えば、NanoLuc)の再構成に依存している - 大きなBiTまたはLgBiTと呼ばれる大きなもの、17.6kDaのタンパク質、および小さなBiTまたはSmBiTと呼ばれる11個のアミノ酸のみで構成される小さなもの。目的の2つのタンパク質を相補的断片と融合させ、ヒト細胞株中で一過性に発現させる。2つの融合タンパク質が相互作用する場合、細胞透過性基質の添加時にその場で発光が生成される。これら2つの断片は、それらが融合している目的のタンパク質間の相互作用によって一緒にされない限り、それらが最小の親和性で会合するように最適化されている。
一般に、生物発光ベースの方法は、蛍光に基づく方法よりもいくつかの利点を有する。生物発光シグナルは、バックグラウンド発光がルシフェラーゼ由来のシグナルと比較して無視できる程度であるため、より高いシグナル対ノイズ比を有する16。対照的に、蛍光ベースのアプローチは、通常、自己蛍光の現象によって引き起こされる比較的高いバックグラウンドに苦しむ。その上、生物発光は蛍光よりも分析された細胞にとって有害性が低く、前者の場合のように試料を励起する必要はない。これらの理由から、インビボでPPIを研究するための生物発光アプローチは、フェルスター共鳴エネルギー移動(FRET)や二分子蛍光相補(BiFC)などの一般的に使用される蛍光法よりも競合します。
我々のプロトコールは、目的のタンパク質組み合わせについて得られた発光を、対照の組み合わせについて得られた発光に参照することに依存している。後者は、より大きな断片と融合された試験されたタンパク質の1つと、より小さな断片と融合された対照タンパク質(例えば、HaloTag)とを含む。後者は、哺乳類のタンパク質のいずれとも相互作用するとは予想されない細菌起源のタンパク質である。このタンパク質を対照として使用すると、分析されるゴルジ常駐タンパク質の対のトポロジーに制限が生じる。哺乳動物細胞ではこのタンパク質は細胞質内で合成されるので、目的の両方のタンパク質は少なくとも1つの細胞質尾部を有するべきである。
このアプローチは、PPIの初期スクリーニングに特に有用であり得る。これは、目的の融合タンパク質が、他のアプローチを適用するには単に不十分なレベルで発現される場合に、選択の方法となり得る。同様に、分割ルシフェラーゼ相補アッセイは、目的のタンパク質が高レベルで発現している場合に最良の選択肢となり得るが、これはそれらの細胞内局在化に悪影響を及ぼすか、または非特異的相互作用を強制することが知られている。より小さい断片は11個のアミノ酸しか有さないので、より大きなタグを使用するのが不可能である場合に分割ルシフェラーゼ相補アッセイを適用することができる。最後に、ここで提示する場合のように、他の技術を用いて得られたデータをさらに確認するために採用することができる。

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent mammalian cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td>
			<td>Corning</td>
			<td>356651</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE HD Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td>
			<td>Promega</td>
			<td>GM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium dedicated to the cell line used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT MCS Starter System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td>
		</tr>
		<tr>
			<td>Nano-Glo Live Cell Assay System</td>
			<td>Promega</td>
			<td>N2011</td>
			<td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium, no phenol red</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Oribital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for data analysis (e.g. GraphPad Prism)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

このプロトコルの目的は、ヌクレオチド糖トランスポーター(NST)によって形成される異種複合体を実証するためのスプリットルシフェラーゼ相補の最新の変異体の適用可能性を探索することである。これらのERおよびゴルジ常駐多膜貫通タンパク質は、細胞質的に合成されたヌクレオチド糖を細胞小器官膜全体に運び、グリコシル化を媒介する酵素を基質とともに供給する。NSTは二量体および/またはそれ以上のオリゴマーとして存在する。異なるNST間の異種相互作用も報告されている。この技術がNSTヘテロマー化の現象の研究に適しているかどうかを検証するために、我々は以前にいくつかの他の手段によって会合することが示された2つのゴルジに常駐するNSTの組み合わせに対してそれを試験した。ルシフェラーゼ相補アッセイは、高い発現レベルを必要とせず、しばしばタンパク質の誤局在化を引き起こし、偽陽性のリスクを高めるため、ゴルジ体常在性膜タンパク質間の相互作用の研究に特に適しているようである。

このアプローチで最も信頼性の高いデータを取得するには、考えられるすべての組み合わせをテストする必要があります ( 図 1 参照)。並行して、ポジティブコントロールとネガティブコントロールを含める必要があります。ポジティブコントロールは、相互作用することが知られている2つのタンパク質からなるべきであり、そのうちの1つは大きなフラグメントと融合?...

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

ここで提示されたプロトコルは、スプリットルシフェラーゼ相補アッセイの最新の変異体を用いて、生きた哺乳動物細胞におけるゴルジ常駐III型膜タンパク質と細胞質的に露出したN末端および/またはC末端との間の異種相互作用の発生を実証することを意図している。

スプリットルシフェラーゼ相補アッセイを用いたゴルジレジデントIII型膜タンパク質によって形成される異種複合体の実証

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

demonstration-heterologous-complexes-formed-golgi-resident-type-iii

Research

JoVE Journal

Biology

2.2K ビュー.  University of Wroclaw. ここで提示されたプロトコルは、スプリットルシフェラーゼ相補アッセイの最新の変異体を用いて、生きた哺乳動物細胞におけるゴルジ常駐III型膜タンパク質と細胞質的に露出したN末端および/またはC末端との間の異種相互作用の発生を実証することを意図している。

ビデオ: Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

ルシフェラーゼベースのレポーター遺伝子アッセイで膜の生合成を研究して

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

脂質中間相における膜タンパク質の結晶化

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

タンパク質のメンブレンオーバーレイアッセイ：可溶性および不溶性タンパク質間の相互作用をテストするためのプロトコル in vitroで</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

高分子結晶学による構造決定のための脂質メソフェーズで栽培膜タンパク質の収穫とクライオ冷却結晶

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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 ...