ドイツ科学研究センター(DFG)助成金BE1911/8-1、ロエベプロジェクトGLUE、および膜を越えた輸送と通信共同研究センター(SFB807)の財政支援に感謝します。

<ol>
	<li>Pandey, A., Shin, K., Patterson, R. E., Liu, X. Q., Rainey, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+strategies+for+protein+production+and+purification+enabling+membrane+protein+structural+biology.">Current strategies for protein production and purification enabling membrane protein structural biology.</a> Biochemistry and Cell Biology. 94 (6), 507-527 (2016).</li><li>Ritchie, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+in+phospholipid+bilayer+nanodiscs.">Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.</a> Methods in Enzymology. 464 (09), 211-231 (2009).</li><li>Frauenfeld, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+lipoprotein+nanoparticle+system+for+membrane+proteins.">A novel lipoprotein nanoparticle system for membrane proteins.</a> Nature Methods. 13 (4), 345-351 (2016).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+stability+can+be+compromised+by+detergent+interactions+with+the+extramembranous+soluble+domains.">Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.</a> Protein Science. 23 (6), 769-789 (2014).</li><li>Seddon, A. 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CF発現とナノディスクへの機能性MPの共翻訳挿入を最適化するためのセットアップと戦略について説明します。必要な機器は、バイオリアクター、フレンチプレス装置など、UV/VISおよび蛍光リーダー、2コンパートメント構成のセットアップに適したCF反応容器、および温度制御されたインキュベーターで構成されます。さらに標準的な装置は、大腸菌細胞を回収するための遠心分離機と...

膜タンパク質(MP)は、水性環境に不溶であるため、タンパク質生産研究において困難な標的です。従来のMP生産プラットフォームは、 大腸菌、酵母、真核細胞などの細胞ベースのシステムで構成されています。合成された組換えMPは、細胞膜から抽出されるか、封入体1からリフォールディングされます。界面活性剤の可溶化後、MPは、確立されたin vitro再構成プロトコルによって適切な膜環境に移すことができます。小胞およびリポソームに加えて、MPはナノディスク2 またはサリプロ3 粒子の形態の平面膜に再構成され、最近では日常的な技術となっている。ただし、これらの戦略はすべて、MPとの界面活性剤の接触を意味し、不安定化、オリゴマーの解離、さらにはタンパク質の構造と活性の喪失を引き起こす可能性があります4。したがって、最適な洗剤の可溶化および再構成条件のスクリーニングは、面倒で時間がかかる場合があります5。
無細胞(CF)系の開放性により、発現反応は、定義された脂質組成を有する予め形成された膜で直接供給される。この脂質ベースの発現モード(L-CF)では、合成されたMPは、提供された二重層6,7に共翻訳的に挿入する機会を有する(図1)。平面脂質二重層ディスク8を取り囲む膜足場タンパク質(MSP)からなるナノディスクは、この戦略に特に適していると思われる9、10。ナノディスクは、さまざまな脂質を使用してin vitroで日常的に組み立てることができ、非常に安定しており、ストックは最大1 mMまで濃縮できます。しかしながら、大腸菌におけるMSP発現およびその精製は必要である。代替戦略として、MSPは、リポソーム11、12、13と共に供給されるCF反応において標的MPと共に共発現され得る。MSPとMPの両方のDNAテンプレートが反応に添加され、発現時にMP/ナノディスクが形成されます。MSP産生は回避されますが、共発現戦略では最終的なDNAテンプレート濃度の慎重な微調整が必要であり、サンプル産生効率のより高い変動が予想されます。
予め形成されたナノディスクの膜へのMPの共翻訳挿入は、トランスロコン機構およびシグナル配列から独立した非生理学的でまだほとんど知られていないメカニズムである13,14,15,16。挿入効率の主な決定要因は、膜タンパク質の種類と提供された膜の脂質組成であり、負に帯電した脂質が頻繁に好まれます15,17。ナノディスク膜はサイズが比較的限られているため、MP挿入時にかなりの量の脂質が放出されます18。ナノディスクサイズの変動は、より高いオリゴマーMP複合体の挿入および調整を可能にする15,18。とりわけ、ホモオリゴマー複合体の正しい集合は、イオンチャネルKcsA、イオンポンププロテオロドプシン(PR)、および多剤トランスポーターEmrE15,18について示されました。MPは、対称ナノディスク膜に両側から比較的等しい周波数で入る可能性があります。したがって、1つのナノディスクに挿入された異なるモノマーまたはオリゴマーは、反対の配向を有する可能性があることを考慮する必要があります。しかし、配向の偏りは、PR六量体およびKcsA四量体の形成について報告されているように、協同的な挿入機構によって引き起こされる可能性があります18。MP配向のさらなるバイアスは、おそらくナノディスク膜の縁に挿入されたMPの配向スイッチに起因する可能性があります。
大腸菌株からのCFライセートの製造は、信頼できるルーチン技術であり、ほぼすべての生化学実験室で実施することができる19,20。ジスルフィド架橋形成に加えて、MPが大腸菌溶解物を用いて合成される場合、他のほとんどの翻訳後修飾は存在しないと考えられるべきである。これにより、構造研究のためにより均質なサンプルが生成される可能性がありますが、個々のMPターゲットの機能に対する潜在的な影響を排除する必要がある場合があります。しかし、大腸菌CFライセート中のGタンパク質共役受容体(GPCR)14,21,22、真核生物トランスポーター23、または大型ヘテロマーアセンブリ24の高品質サンプルを効率的に生産することは、複雑な標的にも適していることを示しています。サンプルの分取スケール量(≈ 1 mg/mL)は、反応混合物(RM)と供給混合物(FM)コンパートメントで構成される2コンパートメント連続交換セルフリー(CECF)構成で得ることができます。FM体積はRM体積を15〜20倍超え、低分子量エネルギー化合物および前駆体19のリザーバを提供する。したがって、発現反応は数時間延長され、MPターゲットの最終収率が増加します。RMコンパートメントとFMコンパートメントは、10〜14 kDaのカットオフを備えた透析膜によって分離されています。2つのコンパートメントには、CECF反応容器の特別な設計が必要です(図1)。FMを保持するテーラードプレキシガラス容器と組み合わせたRM容器としての市販の透析カセットが好適な例である。MP収量は、RM:FM比を変更するか、一定期間のインキュベーション後にFMを交換することによってさらに操作できます。
MPの収率と品質には、反応パラメータの徹底的な最適化が求められることがよくあります。CF発現の重要な利点は、MPの個々の要件に応じてほぼすべての化合物を修正および微調整できることです。簡単な戦略は、最初に基本的な生産プロトコルを確立してMPの歩留まりを向上させることに焦点を当て、次に反応と折り畳み条件を微調整することによってMPの品質を最適化することです。CFライセートに生理学的プロセスがなく、複雑さが軽減されているため、MP25の効率的な生産の成功率が高くなっています。DNAテンプレートの設計およびMg2+イオン濃度の最適化に関する日常的な考慮事項は、ほとんどの場合、満足のいく収率を得るのに十分である26。発現モードによっては、より多様なパラメータをスクリーニングする必要があるため、MP品質の最適化に時間がかかる場合があります14、17、22。
記載されたCF発現プラットフォームを確立するには、 大腸菌 CFライセート(i)、T7 RNAポリメラーゼ(ii)、ナノディスク(iii)、および塩基性CECF反応化合物(iv)の生産にプロトコルが必要です(図1)。 大腸菌 K12株A1927 またはBL21誘導体は、効率的なS30(30,000 x gでの遠心分離)溶解物の調製に頻繁に使用されます。S30溶解物に加えて、他の g力で遠心分離された対応する溶解物(例えばS12)が使用され得る。ライセートは、発現効率とプロテオームの複雑さが異なります。記載されたプロトコルによって調製されたS30ライセートのプロテオームは、詳細に研究されている28。S30プロテオームには、イオンチャネルの発現および分析時にバックグラウンドの問題を引き起こす可能性のある残留外膜タンパク質がまだ含まれています。このようなターゲットには、S80-S100ライセートの使用が推奨されます。ライセート調製中の貴重な変更は、細胞の対数増殖期半ばでの熱ショックとエタノール供給の組み合わせによるSOS応答の誘導です。得られたHS30ライセートはシャペロンに富んでおり、MPフォールディングを改善するためにS30ライセートとのブレンドで使用することができる22。 大腸菌 ライセートにおけるCF発現は、T7 RNAポリメラーゼ(T7RNAP)によって制御されるプロモーターを含むDNAテンプレートとの共役転写/翻訳プロセスとして操作されます。この酵素は、pAR1219プラスミド29を保有するBL21(DE3)スター細胞で効率的に生産することができます。
MSP1E3D1の2つのコピーは、直径10〜12nmの1つのナノディスクに集合するが、以下に説明するプロトコルは、他のMSP誘導体に対しても機能する可能性がある。ただし、MSP1E3D1で形成されたナノディスクよりも大きいナノディスクは安定性が低下する傾向があり、MSP1などのMSP誘導体で形成された小さなナノディスクは、より大きなMPまたはMP複合体に対応できない可能性があります。MSP1E3D1ナノディスクは、多種多様な異なる脂質または脂質混合物を用いてin vitroで組み立てることができます。予め形成されたナノディスクは通常、-80°Cで>1年間安定ですが、安定性は脂質成分によって異なる場合があります。MPの折り畳みと安定性に対する脂質効果のスクリーニングのためには、代表的な種類の脂質/脂質混合物で組み立てられたナノディスクの包括的なセットを準備する必要があります。1,2-ジミリストイル-sn-グリセロ-3-ホスホ-(1'-rac-グリセロール)(DMPG)、1,2-ジミリストイル-sn-グリセロ-3-ホスホコリン(DMPC)、1,2-ジオレオイル-sn-グリセロ-3-ホスホ-rac-(1-グリセロール)(DOPG)、1,2-ジオレオイル-sn-グリセロ-3-ホスホコリン(DOPC)、1-パルミトイル-2-オレオイル-sn-グリセロ-3-ホスホ-(1'-rac-グリセロール)(POPG)および1-パルミトイル-2-オレオイル-グリセロ-3-ホスホコリン(POPC)。
3 mL CECF反応を調製するためのプロトコルについて説明します。1:1の比率でさらに拡大または縮小することが可能です。2コンパートメントCECF構成の場合、すべての化合物を含むRMと低分子量化合物のみを含むFMを準備する必要があります。10〜14 kDa MWCOの市販の3 mL透析装置はRM容器として使用でき、RM容器はFMを保持するカスタムメイドのプレキシガラス容器に入れられます(図1D)30。RM:FMの比率は1:20であるため、3 mL RMに対して60 mLのFMを準備する必要があります。 いくつかの成分の品質、濃度、または種類は、合成されたMPの最終的な収率および/または品質にとって重要な場合があります(表1)。DNAテンプレートは、公表されたガイドラインに従って調製されるべきであり、ターゲットのリーディングフレームのコドン最適化は、生成物収率をさらに有意に改善することができる26。分取スケールCECF反応について、PRの産生のための確立されたプロトコルが記載されている。新しいMPの発現プロトコルを確立するには、通常、収率と品質を向上させるために、特定の化合物の最適化スクリーニングを実行する必要があります(表1)。Mg2+ イオンについては、異なるDNAテンプレートに対して有意な変動を頻繁に示す、焦点を絞った最適な濃度が存在する。T7RNAPまたはS30ライセートの新しいバッチなどの他のCF反応化合物は、最適なMg2+ 濃度をさらにシフトさせる可能性があります。一例として、14〜24mMの濃度の範囲内でかつ2mMのステップで典型的なMg2+ スクリーンが説明される。各濃度は、二重および分析スケールのCECF反応でスクリーニングされます。CECF反応容器として、RMを保持するカスタムメイドのMini−CECFプレキシグラス容器30 が、FMを保持する標準的な24ウェルマイクロプレートと組み合わせて使用される(図1B)。あるいは、96深さウェルマイクロプレートまたは他の市販の透析装置と組み合わせた市販の透析カートリッジを適切なセットアップで使用してもよい(図1C)。

<table>
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (DMPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840445P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850345C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840475C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (POPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)</td>
			<td>Carl Roth (Germany)</td>
			<td>4855</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>4227</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth (Germany)</td>
			<td>9781</td>
			<td></td>
		</tr>
		<tr>
			<td>[3H]-dihydroalprenolol Hydrochloride</td>
			<td>American Radiolabeled Chemicals (USA)</td>
			<td>ART0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl phosphate lithium potassium salt (ACP)</td>
			<td>Merck (Germany)</td>
			<td>1409</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate (ATP)</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr>
			<td>Alprenolol hydrochloride</td>
			<td>Merck (Germany)</td>
			<td>A0360000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange chromatography column material: Q-sepharose&#174;</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>Q1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Type GE 446EC-1</td>
			<td>Gettinge (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor Type 884 124/1</td>
			<td>B.Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid</td>
			<td>Carl Roth (Germany)</td>
			<td>8137</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R250</td>
			<td>Carl Roth (Germany)</td>
			<td>3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks 500 ml baffled flasks, 2 l baffled flasks</td>
			<td>Schott Duran (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate disodium salt</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>C1506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose monohydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6780</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassiumhydrogen phosphate trihydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing SpectrumTM Labs Spectra/PorTM 12-14 kD MWCO Standard RC tubing</td>
			<td>Fisher Scientific (Germany)</td>
			<td>8700152</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreit</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell disruptor</td>
			<td>SLM; Amico Instruments (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth (Germany)</td>
			<td>3783</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate di-sodium salt (GTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>K025</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAC column: HiTrap IMAC HP 5 mL</td>
			<td>GE Life Sciences (Germany)</td>
			<td>GE17-5248</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth (Germany)</td>
			<td>3899</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td>Carl Roth (Germany)</td>
			<td>2316</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Carl Roth (Germany)</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Carl Roth (Germany)</td>
			<td>3076.1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Carl Roth (Germany)</td>
			<td>HN23</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>T202</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Carl Roth (Germany)</td>
			<td>T203</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>3774</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Carl Roth (Germany)</td>
			<td>3772</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine</td>
			<td>Carl Roth (Germany)</td>
			<td>3187</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Carl Roth (Germany)</td>
			<td>3852</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Carl Roth (Germany)</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Carl Roth (Germany)</td>
			<td>1699</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Carl Roth (Germany)</td>
			<td>4207</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Carl Roth (Germany)</td>
			<td>9359</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Carl Roth (Germany)</td>
			<td>1713</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Carl Roth (Germany)</td>
			<td>1709</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Carl Roth (Germany)</td>
			<td>4682</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Carl Roth (Germany)</td>
			<td>1738</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophane</td>
			<td>Carl Roth (Germany)</td>
			<td>7700</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Carl Roth (Germany)</td>
			<td>T207</td>
			<td></td>
		</tr>
		<tr>
			<td>MD100 dialysis units</td>
			<td>Scienova (Germany)</td>
			<td>40077</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2-Hydroxyethylpiperazine-N&#39;-2-ethansulfonic acid (HEPES)</td>
			<td>Carl Roth (Germany)</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecylphosphocholine</td>
			<td>Antrace (USA)</td>
			<td>F308S</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE chamber: Mini-Protean Tetra Cell</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel casting system: Mini Protean Handcast systems</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel power supply: Power Pac 3000</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone/peptone from caseine</td>
			<td>Carl Roth (Germany)</td>
			<td>6681</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump: ip-12</td>
			<td>Ismatec (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenol pyruvate monopotassium salt</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>860077</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth (Germany)</td>
			<td>P018</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Carl Roth (Germany)</td>
			<td>4986</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate Kinase</td>
			<td>Roche (Germany)</td>
			<td>10109045001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter: Hidex 300 SL</td>
			<td>Hidex (Finland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth (Germany)</td>
			<td>8029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>K305</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>P029</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Nanodrop</td>
			<td>Peqlab (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer/fluorescence reader Spark&#174;</td>
			<td>Tecan (Switzerland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (E. coli)</td>
			<td>Roche (Germany)</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra sonificator</td>
			<td>Labsonic U, B. Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#8217;-triphosphate tri-sodium salt (UTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>U6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-30 antifoam</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>A6457</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Carl Roth (Germany)</td>
			<td>2904</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-translational insertion of membrane proteins into preformed nanodiscs

無細胞発現システムにより、膜タンパク質などの複雑なタンパク質の機能的フォールディングをサポートするために、反応環境を調整して設計することができます。ナノディスクとして供給される事前に形成され定義された膜への膜タンパク質の共翻訳挿入および折り畳みの実験手順が実証されています。このプロトコルは完全に洗剤を含まず、1日以内にミリグラムの精製サンプルを生成できます。得られた膜タンパク質/ナノディスクサンプルは、結晶化、核磁気共鳴、電子顕微鏡などのさまざまな機能研究や構造応用に使用できます。無細胞溶解物、設計されたメンブレンを備えたナノディスク、重要なストック溶液、および2コンパートメント無細胞発現反応の組み立てなどの基本的な主要コンポーネントの調製について説明します。膜タンパク質のフォールディング要件は非常に多様であるため、このプロトコルの主な焦点は、重要な塩基性反応化合物、ナノディスクの膜組成、酸化還元およびシャペロン環境、DNAテンプレート設計など、サンプル品質にとって重要なパラメーターと反応ステップの調節です。全過程は、プロテオロドプシンおよびGタンパク質共役受容体の合成によって実証される。

合成されたMPの最終的な収率または品質に対する微調整反応化合物の影響が例示されます。頻繁に日常的なアプローチは、システム効率の便利なモニターとして、緑色蛍光タンパク質(GFP)の発現によってCF反応における最適なMg2+濃度を調整することです。一例として、GFPはpET-21a(+)ベクターから14〜24 mMのMg2+濃度で合成されました(図2)。SDS-PAGE分析により、...

Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

予め形成されたナノディスクへの共翻訳挿入により、界面活性剤と接触することなく、定義された脂質環境で無細胞合成膜タンパク質を研究することが可能になります。このプロトコルでは、必須のシステムコンポーネントの調製と、発現効率とサンプル品質を改善するための重要なパラメーターについて説明します。

予め形成されたナノディスクへの膜タンパク質の共翻訳挿入

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

co-translational-insertion-membrane-proteins-into-preformed

Research

JoVE Journal

Biochemistry

3.3K ビュー.  Goethe University Frankfurt. 予め形成されたナノディスクへの共翻訳挿入により、界面活性剤と接触することなく、定義された脂質環境で無細胞合成膜タンパク質を研究することが可能になります。このプロトコルでは、必須のシステムコンポーネントの調製と、発現効率とサンプル品質を改善するための重要なパラメーターについて説明します。

ビデオ: Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

生化学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

透過型電子顕微鏡により膜相互作用タンパク質を視覚化し、分析するための方法

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

窒素キャビテーションと差動遠心分離培養細胞の周辺膜蛋白質の分布を監視することができます。

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

洗剤無料補完充電プロテオリポソーム融合ペプチドを用いた脂質膜に膜タンパク質の超高速再構成。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

洗剤に敏感な膜タンパク質相互作用の検出

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

細胞間の接触でタンパク質間相互作用の分析計蛍光の変動

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

細菌病原体脂肪酸を膜リン脂質に取り込む研究のための鶏卵黄からのリポタンパク質粒子の単離

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

蛍光相撲捕集タンパク質を用いた SUMO 修飾タンパク質の局在化

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick、合理化されたタンパク質結合アッセイのためのバイオチン化ペプチドディスクへの膜タンパク質のワンステップ組み込み

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

脳膜タンパク質のパルミトイル化定量に関するアシル-PEGyl交換ゲルシフトアッセイ

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...