著者は、彼らが競合する経済的利益を持っていないことを宣言。

<ol>
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著者は、ここに記載される精製方法の開発中に博士Mohabir Ramjeesinghの助言を認める。これらの研究は、ヘルスリサーチ（CIHR）および嚢胞性線維症カナダ（CFC）のカナダの研究所からのCEBに営業補助金によってサポートされていました。 PDWEはCIHRとCFCを通じてフェローシップ賞によって部分的にサポートされていました。著者は、嚢胞性線維症財団ピュー（CFFT）による補正、増強博士R·ブリッジズ（医学部のロザリンド大学と科学）から阻害剤化合物および流通の提供を認める。著者はまた、バキュロウイルス系を用いて、重量および変異型CFTRタンパク質を発現するにベイラー細胞培養施設（NIH助成金をP30 CA125123）により優れたサービスを認める。非アクティブなVX-770アナログ、V-09から1188には、バーテックス·ファーマシューティカルズの贈り物だった。

細胞の過剰発現システムの様々な、完全長の、機能的なCFTRの単離のための精製プロトコルの数が限られている。それはWT-CFTRまたはATPアーゼ、平面二重層内の単一チャネル測定を含むチャネル機能の直接測定を含むアッセイにおいて、高機能である適度な量のF508del-とG551D-CFTRの高濃縮の迅速な精製を可能にするように、ここで説明する方法は有利である小分子19-21の研究に関連性の高?...

肺、腸、膵臓および汗腺などの組織における上皮細胞の頂端膜を横切る塩化物輸送は、主に嚢胞性線維症膜コンダクタンス制御因子（CFTR）、ATP-とABCのリン酸化調節性メンバー（ATP-によって媒介される1で概説膜タンパク質のカセット）Cサブファミリー（）を結合。 ABCCサブファミリーの他のメンバーと同様に、CFTRは、単一で、適度なATPアーゼ活性を有し、そのヌクレオチド結合ドメイン（NBDは）の界面に形成された2つのヌクレオチド結合部位にATPが結合する大規模なマルチスパニング膜内在性タンパク質であるサイト。しかし、他のABCCサブファミリーメンバーとは異なり、CFTRは、固有の規制のClチャネルとしてではなく、アクティブな溶質輸送体として発展してきた。 morbiにつながるCFTR原因嚢胞性線維症、肺、消化管、膵臓および生殖器官を含む複数の器官に影響を与える疾患の変異、若年成人におけるdityと死亡。肺疾患は、典型的には、嚢胞性線維症、早期死亡を占めており、ほとんどの場合、誘導気道の表面上皮におけるCFTR機能の喪失によって引き起こされる。繊毛気道上皮の頂端面上に流体層を変更するために表面上皮を横切って水の動き- CFTR塩素イオンチャネル活性の欠如は、両方のClの減少を引き起こす。これは、繊毛呼吸上皮細胞、気道の外に効果的に明確な病原体の能力を損なう粘性の気道表面液が得られる。結果として、ほとんどのCF患者は、肺感染症や炎症による肺損傷の再発発作に苦しむ。 予想されるように、正常なCFTRタンパク質の作用メカニズムの研究は、主に、チャネルゲーティング活動の詳細な電気生理学的研究に焦点を当てた。シングルチャネルの研究では、PKA-依存性Clとして直接そのCFTR機能を示している- ATP規制ゲート2を有しているチャンネル。詳細な電気生理学的研究は、単一のCFTRチャネル1,3上の大量の情報を提供する、しかし研究されており、任意の特定の単一チャネルの特性はCFTRチャネルの全人口、したがって、単一チャネルを反映しているかどうかの懸念があるかもしれ結果は常に巨視的人口を研究するための方法と一緒に考慮されるべきである。精製されたCFTRの人口チャネル活性の直接アッセイは、疾患を引き起こす突然変異と関連する分子の欠陥への洞察を提供し、化学モジュレーター修復変異CFTRタンパク質の発見を駆動する可能性がある。現在までに、嚢胞性線維症4を引き起こすと考えCFTRにおける1900の異なる変異を超えるがある。北米と欧州で患者の約90％の少なくとも一つの対立遺伝子に見られる主要な突然変異、F508del-CFTRは、誤った折り畳みタンパク質につながるND小胞体5における保持。 F508del-CFTRはまた、不良チャネル活性6-9を含む他の結果を持っています。細胞表面からのCFTRの結果として存在しないことは、重篤な疾患と関連している。 G551D-CFTR、あまり一般的変異は、正しくまだ折り畳まれた細胞表面6における塩素イオンチャネルとして機能不全であることが考えられている。小分子補正器及び増強剤の開発は、それぞれ、細胞表面に存在フォールディングおよび/またはそのような細胞表面へF508del-CFTRのような変異体のトラフィッキング、および増強の補正や、G551Dような変異のチャンネル活性を増加させるという目標を有する。補正器VX-809およびVX-661は、（まだ患者での使用が承認されていませんが、増強Kalydeco（ivacaftor、VX-770）は、少なくとも1つのG551Dを持つすべての12 CF患者におけるHR&gt; 6歳150ミリグラムで使用されているG178R、S549N、S549R、G551S、G1244E、S1251N、S1255Pの1の患者のためのより最近-CFTR突然変異、及びとG1349D。 Kalydeco安全かつCF疾患10の臨床的指標の改善をもたらすの両方が、しかし、この小分子の作用機序は十分に患者に使用するためのFDA承認の際に理解している。 CFTRの精製方法の一握りが完了するまでにかなりの時間を必要とする多くは、2,11-18、以前に記載されている。最近の刊行物19には、ユニークな精製および再構成の方法は、S fは 9細胞発現系において過剰発現CFTRについて記載し、定義された脂質系において、この精製されたタンパク質は、CFTRの集団CFTRハロゲンチャネル活性アッセイを開発するために使用された分子。アッセイは、CFTR機能上のリン酸化、ヌクレオチドおよび阻害剤の既知の効果を再現。システムは、重量（野生型）、F508del-及びG551D-CFTRに増強VX-770 / Kalydecoの効果を調べるために使用し、それを最初にするために示された薬物が母集団の観点からCFTRおよびヌクレオチドおよび小分子を有する突然変異体の相互作用の研究にこれらの方法の有用性および適用可能性を実証し、ATP非依存的に、チャネル活性を増強するためにCFTRタンパク質と直接相互作用時間タンパク質約臨床的に関連する質問に答える。方法はまた、他の増強剤分子およびそれらの誘導体20、ならびにタンパク質21の活性に対する小分子の補正の効果を研究するために使用されている。 流出アッセイは、以前に、電極、放射性トレーサー、およびフルオロフォア22,23を用いて、全細胞アッセイは、イオン選択性電極24を有する膜小胞を含むCFTR突然変異体の活性およびその活性に対するCFTR調節性化合物の効果を調査するために多くの研究で使用されている、放射性トレーサー25を用いて再構成CFTRを精製した。しかし目精製された再構成されたCFTRを研究するためのイオン選択電極の電子の使用が最初に最近19報告された。現在の方法の応用は、再構成および緑膿菌 、共通CF病原体における2つの膜タンパク質の機能的特徴付けのために使用されてきた。ヨウ化物流出測定と結合して精製AlgE外膜タンパク質の再構成は、このトランス26を介してアニオン性アルギン酸塩分泌のためのモデルをサポートするために使用された。再構成およびヨウ化物流出測定は、モデルは、このタンパク質27によって細菌の内膜を横切る脂質結合オリゴ糖転位のためのH +依存性アンチポートメカニズムを示唆して提案することが許可され精製された例えば、wzxタンパク質に適用した。どちらの場合も、ヨウ化物は、一つは天然基質のために期待するかもしれないよりも低いスループットではあるが、アニオン性基板の代わりとして使用された。この方法は、cと他のタンパク質への適応のために適切であり得るアニオン性基板のためationic輸送又は伝導経路。 ここで迅速な精製手順は、CFTRタンパク質と定義された脂質のプロテオリポソー​​ムへの再構成のために記載されている。迅速な再構成手順は容易に精製において使用される界面活性剤の種類は、ここで使用される方法による除去に適しているか、再構成手順の前に適切な界面活性剤に交換することができれば、他の方法によって精製CFTRでの使用のために調整することができる。精製された再構成CFTRタンパク質のチャンネル活性を測定するためのヨウ化物流出方法について詳細に説明すると、この方法から得ることができるいくつかの典型的な結果を示す。

<table border="0" cellpadding="0" cellspacing="0" width="1269">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:403px;">fos-choline 14 detergent</td>
			<td style="width:515px;">Anatrace Affymetrix (www.anatrace.affymetrix.com)</td>
			<td style="width:145px;">F312</td>
			<td style="width:207px;">Affymetrix: Anatrace Products; CAS#&#160;:77733-28-9</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>04 693 132 001</td>
			<td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini:1 in 10 mL) (Roche Diagnostics GmbH; Ref: 11873 580 001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&#160;</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>05 892 791 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA Agarose</td>
			<td>Qaigen GmbH</td>
			<td align="right">1018240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand Screening columns</td>
			<td>Fisher Healthcare</td>
			<td>11-387-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal filters, Ultracel-100K</td>
			<td>Millipore (www.millipore.com)</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td>
			<td colspan="2">New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td>
			<td>Peirce PKA is also suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840051C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">POPC</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PS, Porcine Brain</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840032C</td>
			<td>(CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w))</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>841118C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ergosterol</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td align="right">45480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Detergent removal spin column</td>
			<td>Thermo Scientific</td>
			<td align="right">87779</td>
			<td>1 ml capacity columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">valinomycin</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td>V-0627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VX-770 (ivacaftor)</td>
			<td>Selleck Chemicals</td>
			<td>S1144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iodide selective microelectrode</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td>
			<td>LIS-146ICM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clampex 8.1 software</td>
			<td>Axon Instruments (www.axon.com)</td>
			<td></td>
			<td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alternate Software: ArrowLabb System&#160;</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td>
			<td>LIS-146LICM-XS</td>
			<td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&#160; This software should serve a similar function to our setup</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">small stir bars</td>
			<td>Big Science Inc (www.stirbars.com)</td>
			<td>SBM-0502-CMB</td>
			<td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sephadex G50, fine</td>
			<td>GE Health Care (www.gelifesciences.com)</td>
			<td>17-0042-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Laboratory Supplies Co, Inc.</td>
			<td>G112SP1G</td>
			<td>Bath sonicators from other manufacturers should also be suitable</td>
		</tr>
	</tbody>
</table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

嚢胞性線維症膜コンダクタンス制御因子（CFTR）の輸送体のATP結合カセット（ABC）スーパーファミリーのユニークな流路形成部材である。 CFTRのリン酸化およびヌクレオチド依存性クロライドチャネル活性は、しばしば全細胞系および切除した膜パッチの単一チャネルとして研究されている。多くの嚢胞性線維症の原因となる変異はこの活性を変化させることが示されている。精製プロトコールの少数が公開されているが、チャネルの活性を保持する高速再構成方法及び精製系人口チャネル活性を研究するための適切な方法は、欠けている。ここで迅速な方法は、調整されたハロゲン化チャネルとしての活性を保持して定義された脂質組成物のプロテオリポソー​​ムに精製および全長CFTRタンパク質の機能的再構成のために記載されている。一緒にチャンネル活性の新規のフラックスベースのアッセイと、この再構成方法は、適したシステムである野生型CFTRと病気の原因となる変異体F508del-とG551D-CFTRの人口チャネル特性を研究。具体的には、本方法は、リン酸化、ヌクレオチドおよびそのようなCFTRチャネル活性における増強および阻害剤としての小分子の直接的な効果の研究において有用性を有する。方法はまた、アニオン性基材用の他の膜チャネル/トランスポーターの研究に適している。

この書かれた刊行物、精製および再CFTRタンパク質の調節されたチャネル活性を測定するための方法が記載される。 図1aは、精製、再構成およびヨウ化物流出手順のためのワークフローを示している。ヨウ化物フラックスによる再構成およびチャンネル活性測定のための方法は、関連するビデオにさらに詳細に示されている。 プロテオへのCFTRの精製と再構成<...

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

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

ここで説明すると、嚢胞性線維症における欠陥があるこの塩素チャネル、のための活動を維持して精製野生型および変異型CFTRタンパク質の機能的再構成するための迅速かつ効果的な手続きである。 CFTRによって媒介され、再構成プロテオリポソー​​ムからヨウ化物流出は、チャネル活性の研究や小分子の効果を可能にする。

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

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

functional-reconstitution-channel-activity-measurements-purified

Research

JoVE Journal

Biology

12.1K ビュー.  Hospital for Sick Children. ここで説明すると、嚢胞性線維症における欠陥があるこの塩素チャネル、のための活動を維持して精製野生型および変異型CFTRタンパク質の機能的再構成するための迅速かつ効果的な手続きである。 CFTRによって媒介され、再構成プロテオリポソー​​ムからヨウ化物流出は、チャネル活性の研究や小分子の効果を可能にする。 

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

生物学

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

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

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

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

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

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

ステロイド受容体の生化学的再構成には、•Hsp90のタンパク質複合体とリガンド結合の再活性化

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

リジン脱メチル化酵素活性の定量測定のためのMassSQUIRMのアプリケーション

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

上皮細胞ライセートからのGSTタグ付き突然変異体のRhoタンパク質（GST-RhoAは（G17A））を使用してアクティブロー-GEFの親和性降水

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-依存性のCFTRの高分子シグナル伝達複合体のin vitroでの解析で

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

構造と機能研究のための脂質膜へのKvチャネルの再構築

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

植物や緑藻の光捕集複合体のin vitro再構成では、

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

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 ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

膜貫通タンパク質の再構成、顕微鏡およびパッチクランプ研究のための巨大単膜小胞への電位依存性イオンチャネル、KvAP、

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...