この作品は、保健研究のカナダの研究所によってサポートされていました。 CSCは、自然科学とカナダの工学研究評議会からのポスドクによって賄われていた。 FDはティアIIカナダリサーチチェアです。

<html>全体の再構築プロセス再構成プロセスは、洗剤で可溶化したリン脂質の存在下で精製MalFGK 2複合体と膜骨格タンパク質（MSP）を混合することによって開始されます。ステップは、Bio-ビーズまたはアンバーライト（ 図1）と呼ばれるポリスチレン系吸着材による洗剤の遅い除去のあとに続いています。自動組立工程が原因疎水性リン脂質、MalFGK 2錯体とMSP両親媒性タンパク質の表面との間の非極性相互作用の可能性が高くなります。最終製品はMalFGK 2錯体を包み込むMSPの2分子からなる円盤状の粒子である。粒子は、超遠心分離および分析用サイズ排除クロマトグラフィーにより付加物および凝集体から分離される。粒子はネイティブゲル電気泳動および動的光散乱分光法によって特徴付けられる。 膜足場タンパク質、MSPのタイトル &quot;&gt; 1。準備<ol><li> hisタグMSPは、（バージョンMSP1D1 1） 大腸菌におけるプラスミドpMSP1D1から生産され大腸菌 BL21（DE3）OD 600に誘起〜0.5mMのイソプロピル-β-D-1-チオガラクトピラノシドを37℃で3時間℃で0.5 </li><li>細胞は4℃で10分間5000×gで℃で遠心分離により回収し、100μMフェニルメタンスルホニルフッ化物を含むTSG10バッファーに再懸濁する。 </li><li>セルが8000 psiでフレンチプレス（3×）で溶解させ、不溶物を4℃で10分間5000 xgで遠心分離により除去されるMSPを含む可溶性画分を45分間125000×gで超遠心分離によって分離されています。 </li><li> MSPは、ニッケルキレートクロマトグラフィーTSG10バッファに〜1.5ミリリットルNiセファロースHPを用いて精製する。汚染物質を、50mMイミダゾールを含むTSG10バッファーで洗い流される。 MSPは600 mMを含むTSG10緩衝液で溶出されイミダゾール。精製したタンパク質はTSG10バッファに透析し、10月15日〜mg / mlのタンパク質濃度で-70℃に保存されています。 </li></ol> 2。 MalFGK 2複合体の調製<ol><li> MalFGK 2複合体は、MalKのC末端にHisタグ、 大腸菌におけるプラスミドpBAD22-FGKから発現される大腸菌 BL21（DE3）およびOD 600に誘起〜37℃で3時間で0.2％のL-アラビノース℃で0.5 </li><li>ステップ1.3のように、細胞溶解及び遠心分離後、膜ペレットを5 mg / mlの最終濃度でTSG20バッファーに再懸濁する。材料は、穏やかに振盪しながら4℃で3時間1％w / vのn-ドデシル-β-マルトピラノシド（DDM）℃で可溶化する。 </li><li>不溶性物質は、ステップ1.3および可溶化を含む上清のように超遠心分離により除去される MalFGK 2複合体を回収し、精製工程1.4のように、しかし、透析せずにされています。 </li><li> Furtherの浄化をSuperdex 200 HR 10 / 0.5ml /分の流速で300カラムでTSGDバッファーでゲルろ過クロマトグラフィーによって達成される。 </li></ol> 3。リン脂質の調製<ol><li> E.クロロホルムに溶解させた大腸菌総脂質抽出物は、スクリューキャップマイクロチューブは1,000 nmolのアリコートに分けられます。溶媒を窒素気流下で蒸発させ、真空デシケーター中でさらに一晩乾燥させる。 </li><li>脂質膜は、5 nMの最終濃度でTSバッファに溶解し、激しくボルテックスや超音波処理する。溶解した脂質が原因で水性TSバッファにおける彼らの一般的な不溶性に若干不透明に見えるでしょうが、彼らは、懸濁液のままにしてください。 DDMは、溶液が透明になる時0.5％（〜10mm）の最終濃度に添加される。 </li><li>脂質混合物を約5秒間5回超音波処理し、水浴及びパルスに配置されます。脂質混合物を1おしっこの最大4℃で保存されているK。 </li></ol> 4。バイオビーズの調製<ol><li>約10〜15ミリリットル（乾燥体積）バイオビーズを50mlチューブに入れています。 </li><li>ビーズを順次100％メタノール、95％エタノール、ミリQ H 2 O、および最終的にTSバッファ（二回）50mlで洗浄されています。 </li><li>洗浄したビーズは、4℃で保存するC〜10ミリリットルTSバッファインチ</li></ol> 5。 Nanodisc再構成<ol><li> MSPは〜7 mg / mlの（〜0.3 mM）の最終濃度でTSGD緩衝液中で希釈されています。 </li><li> MSP：TSGDバッファ内1:3:60または1:3:400の脂質比精製MalFGK 2複合体の約2 nmolのはタンパク質で混合される。最終濃度は6μmMalFGK 2、18μMのMSPおよび360μMの脂質（1:3:60）または2.4 mMの脂質（1:3:400）です。最終容量は300μlです。最終DDMの濃度は0.08％（〜1.6 mm）です。グリセロールの最終濃度は脂質の量に依存する追加されましたが、周りに5から10パーセントV / Vのまま</lI&gt; <li> 〜バイオビーズ懸濁液50μlをチューブに添加し、混合物を4℃で揺動テーブルの上に一晩インキュベートする。 </li><li>ビーズは、重力により沈降し、溶液を、できるだけ多くのバイオビーズを避けるために、狭い先端から分注される。 </li><li>沈殿大は20分間100,000×gで超遠心分離により除去される。ディスクはTSG10バッファ内のステップ2.4​​のようにゲルろ過クロマトグラフィーにより精製される。ディスクを含む画分をプールし、アリコート製剤の安定性をテストするために、同じゲル濾過カラムに再注入する必要があります。 </li><li>精製されたディスクは、長時間にわたって-70℃で保存することができます。氷の上で解凍後、ディスクは潜在的な沈殿物を除去するために、ステップ5.5のように、超遠心分離を行うべきである。アリコートは有意な凝集や沈殿が解凍時に発生しなかったことを確認するためにゲルろ過クロマトグラフィーで分析する必要がありますプロセス。 </li></ol> 6。ネイティブゲル電気泳動<ol><li> nanodiscsの1μM（〜0.2㎎/ ml）を再構成する（ 図3A）の品質を評価するためのネイティブPAGE 6,7（トリス-HCl pH 8.8、4-12％）によって分析される。 </li><li> 2複雑なMalFGKに男性の結合（1μM）はnanodiscsで再構成はまた、未変性PAGE（ 図3A）により評価される。 </li><li>泳動後、ゲルを10分間クマシーブルーで染色されており、約1時間脱色した。 </li></ol> 7。動的光散乱（DLS） <ol><li> Nanodiscsを​​Superdex 200 HR 10 / 0.1ml /分の流速で300カラムを用いてTSG10バッファ内のステップ2.4​​のように、ゲルろ過クロマトグラフィーにより精製される。 nanodiscsを​​含む画分をプールし、アミコン遠心フィルターで〜10 mg / mlに集中している。 </li><li>サンプルは使用DLSによって分析前（0.22μmフィルター）2回濾過さ1μlの内部体積石英キュベットでDynaPro nanostar楽器（ワイアットテクノロジー）。データは直径、粒子の分子量を推定するためにDynamicsソフトウェア（ワイアット·テクノロジー）を使用して装着されている。 </li></ol> 8。 ATPase活性測定<ol><li> MalFGK 2 -再構成nanodiscsのATPase活性は比色分析8を用いて決定される。 </li><li>精製されたディスクと1mMのATP1μMのは20分間、男性と37℃での増加量と一緒に混合されています。無機リン酸塩の放出は660 nmで測定する。 </li><li>解放リン酸の量は、リン標準溶液を用いて作成した標準曲線と比較されます。 </li></ol> 9。代表的な結果 nanodiscsは、ゲルろ過クロマトグラフィー（ 図2A、左 ）によって精製される。クロマトグラムを示し、その再構成されたdの過半数ISCS（黒のトレース）の単一ピークとして溶出し、一方、過剰な脂質（赤いトレース）ボイドボリュームに溶出して、広範な一連のピークとして作らディスク。 nanodiscsの質はさらに、ネイティブゲル電気泳動および動的光散乱法（DLS）によって分析されます。脂質の過剰の存在下で再構成されたものはスミア（ 図2A、右 ）のように移行するのに対し、適切に再構成されたディスクには、ゲル上の鋭いバンドとして移行されます。 DLSによる分析は、ディスクの人口は11.4 nmの（ 図2B）の平均粒径が均一であることを示しています。再構成されたディスクはDLS近似に基づいた215 kDaの見かけの分子量を有する。過剰な脂質の存在下で再構成したサンプルは、非均質な試料のために典型的である100nmであり、周囲に広く分布している半径を表示します。 MalFGK 2複合体の質がATPアーゼによるネイティブ·ゲル電気泳動し、その活性によって評価される測定結果（ 図3）。マルトース結合タンパク質の男性はMalFGK 2輸送9,10への高親和性で結合する。非変性ゲル電気泳動を使用して、それは男性とMalFGK 2（ 図3A）との複合体を検出することが可能である。男性がMalK 2 ATPase活性の刺激は、 図3Bに示されている。 <img src="/files/ftp_upload/3910/3910fig1.jpg" alt="図1"/> 図1。再構成プロトコルのための典型的なフローチャート。 <img src="/files/ftp_upload/3910/3910fig2.jpg" alt="図2"/> 図2。 nanodisc準備。Aの品質管理または高脂質比（1/3/400、赤いトレース）; nanodiscsのゲル濾過分析（スーパーデックス200 HR 10/300カラム）は、低脂質比（黒トレース1/3/60）で再構成した。同じディスクの準備のネイティブゲル電気泳動。モルkDaでecular量マーカーが表示されている。同じディスクの準備B.動的光散乱分析。 <a href="http://www.jove.com/files/ftp_upload/3910/3910fig2large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/3910/3910fig3.jpg" alt="図3"/> 図3。 MalFGK 2 nanodisc粒子の分析。A. MalFGK 2 nanodisc粒子の量が増加するとともにインキュベート男性のゲルシフト解析。男性の濃度の関数としてMalFGK 2 nanodisc粒子BのATPase活性。

<ol>
	<li>Denisov, I. G., Ginkova, Y. V., Lazarides, A. A., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+monodisperse+phospholipid+bilayer+Nanodiscs+with+controlled+size.">Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.</a> J. Am. Chem. Soc. 126, 3477-3487 (2004).</li><li>Boldog, T., Grimme, S., Li, M., Sligar, S. G., Hazelbauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+separate+chemoreceptor+oligomeric+states+and+reveal+their+signaling+properties.">Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.</a> Proc. Natl. Acad. Sci. USA. 103, 11509-11514 (2006).</li><li>Bass, B. J., Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homotropic+cooperativity+of+monomeric+cytochrome+P450+3A4+in+a+nanoscale+native+bilayer+environment.">Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.</a> J. Biol. Chem. 282, 7066-7076 (2007).</li><li>Alami, M., Dalal, K., Lelj-Garolla, B., Sligar, S. G., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+unravel+the+interaction+between+the+SecYEG+channel+and+its+cytosolic+partner+SecA.">Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.</a> EMBO J. 26, 1995-2004 (2007).</li><li>Mi, L. -. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+stability+of+the+epidermal+growth+factor+receptor+in+detergent+micelles+and+phospholipid+nanodiscs.">Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.</a> Biochemistry. 47, 10314-10323 (2008).</li><li>Sch&#228;gger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal. Biochem. 217, 220-230 (1994).</li><li>Dalal, K., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+SecY+translocon+in+Nanodiscs.">Reconstitution of the SecY translocon in Nanodiscs.</a> Methods Mol. Biol. 619, 145-156 (2010).</li><li>Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., Candia, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+assay+for+nanomole+amounts+of+inorganic+phosphate.">An improved assay for nanomole amounts of inorganic phosphate.</a> Anal. Biochem. 100, 95-97 (1979).</li><li>Davidson, A. L., Dassa, E., Orelle, C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+evolution+of+bacterial+ATP-binding+cassette+systems.">Structure, function and evolution of bacterial ATP-binding cassette systems.</a> Microbiol. Mol. Biol. Rev. 72, 317-364 (2008).</li><li>Bordignon, E., Grote, M., Schneider, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maltose+ATP-binding+cassette+transporter+in+the+21st+century-towards+a+structural+dynamic+perspective+on+its+mode+of+action.">The maltose ATP-binding cassette transporter in the 21st century-towards a structural dynamic perspective on its mode of action.</a> Mol. Microbiol. 77, 1354-1366 (2010).</li><li>Alvarez, F. J., Orelle, C., Davidson, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+an+ABC+transporter+for+use+in+electron+paramagnetic+resonance+spectroscopy.">Functional reconstitution of an ABC transporter for use in electron paramagnetic resonance spectroscopy.</a> J. Am. Chem. Soc. 132, 9513-9515 (2010).</li><li>Ritchie, T. K., Grinkova, Y. V., Bayburt, T. H., Denisov, I. G., Zolnerciks, J. K., Atkins, W. M., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+Nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 464, 211-231 (2009).</li><li>Gl&#252;ck, J. M., Koenig, B. W., Willbold, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+allow+the+use+of+integral+membrane+proteins+as+analytes+in+surface+plasmon+resonance+studies.">Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies.</a> Anal. Biochem. 408, 46-52 (2011).</li><li>Wan, C. -. P. L., Chiu, M. H., Wu, X., Lee, S. K., Prenner, E. J., Weers, P. M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein-induced+conversion+of+phosphatidylcholine+bilayer+vesicles+into+nanodisks.">Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks.</a> Biochim. Biophys. Acta (BBA). 1808, 606-613 (2011).</li><li>Nath, A., Trexler, A. J., Koo, P. K., Miranker, A. D., Atkins, W. M., Rhoades, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+spectroscopy+using+phospholipid+bilayer+Nanodiscs.">Single-molecule fluorescence spectroscopy using phospholipid bilayer Nanodiscs.</a> Methods Enzymol. 472, 89-117 (2010).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochromes+P450+in+Nanodiscs.">Cytochromes P450 in Nanodiscs.</a> Biochim. Biophys. Acta. 1814, 223-229 (2011).</li><li>Zhang, X. X., Chan, C. S., Bao, H., Fang, Y., Foster, L. J., Duong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+and+SILAC-based+mass+spectrometry+to+identify+a+membrane+protein+interactome.">Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome.</a> J. Proteome Res. , (2011).</li></ol>

特別な利害関係は宣言されません。

我々はnanodiscsにマルトーストランスポーターの再構成のための簡単​​な手順を説明します。トランスポーターは、ATPアーゼ活性であり、水溶性の結合パートナーの男性との相互作用が（ 図3）再現することができます。 nanodiscsにトランスポーターの成功した再構成には、追加の生物物理学的および生化学的解析のための道を開く。特に興味深いのは、体系的な分析洗剤、リポ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>アミコンウルトラ-4 50K遠心フィルター</td><td>ミリポア</td><td> UFC805008 </td><td>適切な使用については、製造元のプロトコルに従ってください</td></tr><tr><td>バイオビーズSM-2吸着剤</td><td>バイオ·ラッド</td><td> 152-3920 </td><td></td></tr><tr><td> 大腸菌総脂質</td><td>アヴァンティ極性脂質</td><td> 100500C </td><td>クロロホルムに溶解し、有機溶剤に応じて扱う</td></tr><tr><td> NiセファロースHPの樹脂</td><td> GEヘルスケア</td><td> 17-5268-01 </td><td></td></tr><tr><td>リン標準溶液</td><td>シグマアルドリッチ</td><td> P3869 </td><td></td></tr><tr><td> pMSP1D1 </td><td> Addgene </td><td> 20061 </td><td></td></tr><tr><td>たSuperdex 200 HR 300分の10 </td><td> GEヘルスケア</td><td> 17-5172-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表Iに特異的な試薬。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 名前 </td><td> 構図 </td><td> 注釈 </td></tr><tr><td> DDMの株式</td><td>の10％w / vのDDM </td><td> -20℃でミリQ水とストアに再懸濁</td></tr><tr><td> MalFGK 2株式</td><td> 1から2 mg / mlの 50mMトリス-HCl、pH7.9 100mMのNaCl 10％v / vのグリセロール 0.03パーセントw / vのDDM </td><td> -70℃で保存精製後</td></tr><tr><td> MSPの株式</td><td> 10から15 mg / mlの 50mMトリス-HCl、pH7.9 100mMのNaCl 10％v / vのグリセロール</td><td> -70℃で保存&lt;1mlのアリコートで精製後や過度の凍結/融解サイクルを避ける</td></tr><tr><td>リン脂質株式</td><td> 5nMのE.大腸菌総脂質 0.5パーセントw / vの（10mM）を、DDM 50mMのトリス-HCl、pH 7.9 50mMのNaCl </td><td> 1週間に4℃で保存</td></tr><tr><td> TSバッファ</td><td> 50mMのトリス-HCl、pH 7.9 50mMのNaCl </td><td> 4℃で保存</td></tr><tr><td> TSG10バッファ</td><td> 50mMトリス-HCl、pH7.9 100mMのNaCl 10％v / vのグリセロール</td><td> 4℃で保存</td></tr><tr><td> TSG20バッファ</td><td> 50mMトリス-HCl、PH8 100mMのNaCl 20パーセントv / vグリセロール</td><td> 4℃で保存</td></tr><tr><td> TSGDバッファ</td><td> 50mMトリス-HCl、pH7.9 100mMのNaCl 10％v / vのグリセロール 0.03パーセントw / vのDDM </td><td> 4℃で保存し、使用直前にDDMを追加</td></tr><tr><td colspan="3"> 表II。ソリューションレシピ。 </td></tr></tbody></table></td></tr></tbody></table>

a step-by-step method for the reconstitution of an abc transporter into nanodisc lipid particles

nanodiscは、円盤状の粒子（〜10月12日NM大）リン脂質二重層の小さなパッチにトラップ膜タンパク質ということです。 nanodiscは特にリガンド - 受容体相互作用の文脈では、膜タンパク質を研究するために特に魅力的な選択肢である。 Sligarらによって開拓方法はアポリポタンパク質A1由来組み換え高ヘリカル足場タンパク質の両親媒性の性質に基づいている。極地は、水性環境に直面し、一方足場タンパク質の疎水性面は、脂質二重層の脂肪アシル側鎖と相互作用する。粒子が小さく、均質かつ水溶性であるためnanodiscsにおける膜タンパク質の解析がリポソーム上の重要な利点を持っています。また、通常は水溶性タンパク質に予約生化学的および生物物理学的手法を適用し、膜のいずれかの側からすることができます。この視覚的なプロトコルでは、我々はよく特性のステップ·バイ·ステップの再構成を提示terized細菌のABCトランスポーター、男性MalFGK 2複合体。ディスクの形成は、洗剤の進歩的な除去の際に行われている疎水性相互作用に依存して自己組織化プロセスである。私たちは本質的な手順を説明し、私たちは集合体と大きい多分散リポソーム状粒子の形成を制限するために、正しいタンパク質対脂質比を選択することの重要性を強調。単純な品質は、そのようなディスクが正しく再構成されていることを確認し、ゲル濾過クロマトグラフィー、天然ゲル電気泳動および動的光散乱分光法として制御します。

Watch this Scientific Journal Video about A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles at JoVE.com

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Nanodiscsはリン脂質二重層の小さなパッチに膜タンパク質を組み込んだ小さな円盤状の粒子である。我々は、ディスクへのMalFGK2ポーターのステップ·バイ·ステップの取り込みを示し視覚プロトコルを提供します。

Nanodisc脂質粒子にABCトランスポーターの再構成のためのステップ·バイ·ステップ方式

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

a-step-step-method-for-reconstitution-an-abc-transporter-into

Research

JoVE Journal

Biology

25.3K ビュー.  University of British Columbia. Nanodiscsはリン脂質二重層の小さなパッチに膜タンパク質を組み込んだ小さな円盤状の粒子である。我々は、ディスクへのMalFGK2ポーターのステップ·バイ·ステップの取り込みを示し視覚プロトコルを提供します。

ビデオ: A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表現、界面活性剤可溶化、および膜トランスポーター、MexBの多剤耐性タンパク質の精製

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

生物学

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

イメージングのための簡単​​な方法シロイヌナズナ浸潤イメージング媒体としてPerfluorodecalinを使って葉

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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 ...

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 ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

植物抽出物からの分散粒子の除去のための凝集剤の効率を評価するための手順

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

統合欠陥レンチウイルスベクターにパッケージ化された転写調節睡眠美容システムによって効率的な体外転位法

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

毒性試験のための空気中の粒子への現実的な反復吸入暴露のための気管支上皮モデル

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...