この作品は、NIH (RO1 NS090390) によって支えられました。博士ジョアン Steffanfor 技術支援とこの試金の開発の間に議論に感謝も申し上げます。

動物の倫理の声明 - 実験が実施されたケアと健康の国民の協会および制度的動物ケアおよび使用委員会 (承認された動物の研究プロトコルの実験動物の使用のためのガイドに従い、IACUC) カリフォルニア大学アーバイン校、AAALAC 認定機関。すべての努力は、動物の苦痛を最小限に抑えるために行われました。
1. 換散バッファーの準備
<ol><li>「水溶性」の換散バッファーの準備 (10 mM Tris pH 7.4 では、1 %x トリトン 100、150 mM の NaCl、10% グリセロール) と 0.22 μ m のフィルターを通して無菌フィルター。<ol>
<li>作業「可溶性」換散バッファーの N-エチルマレイミド (NEM) を測定し、100% に溶解し 100 mM phenylmethysulfonyl フッ (PMSF) で十分に溶かして江藤。 注: 換散バッファー阻害剤: (i) 20 mM N-エチルマレイミド (NEM);(ii) 0.2 mM PMSF;(iii) 1 mM バナジン ナトリウム (Na3VO4);(iv) 1 μ G/ml leupeptin;(v) 1 μ G/ml アプロチニン;(vi) 20 mM フッ化ナトリウム (NaF)。 注意: は、プロトコル全体 NEM を操作するときに、マスクを着用します。換散バッファーの作業行う必要がありますアクティブな阻害剤を確保するため新鮮です。</li>
		<li>プロテアーゼ阻害剤と水溶性の換散バッファーの次の順序で阻害剤を追加: Leupeptin、アプロチニン、バナジン ナトリウム (Na3VO4) 冷たい換散バッファーのボリュームまでフッ化ナトリウム (NaF)。</li>
	</ol>
</li>
	<li>「不溶性」の換散バッファーの「水溶性」換散バッファーの 2 mL に 20% SDS の 500 μ l を追加することにより 4% SDS への補足します。室温に置いたまま。 注: SDS を沈殿が 4 ° C で保管するとき部屋の温度で、これをしてください。最終的な換散バッファー組成: 10 mM トリス (pH 7.4)。1% トリトン X-100;150 mM の NaCl;10% グリセロール;4 %sds (不溶性のみ)。</li>
</ol>2. 水溶性/不溶性分別のプロトコル
注: は、回路図の図 1を参照してください。
<ol><li>各サンプルのため管の 2 つのセットのラベル:「水溶性」と「不溶性」。</li>
	<li>組織に (1.1.1 参照) プロテアーゼ阻害剤を添加した冷たい作業可溶性換散バッファーの X μ L を追加します。 注: X = ボリュームによって異なります組織量;表 1を参照してください。</li>
	<li>マウス脳組織 (すなわち、線条体)、dounce ガラス 1 mL douncer と「不溶性」にピペットでゆっくり 30 回はチューブ (氷の上維持) をラベル付けします。 注: 気泡を形成する不完全な均質化をもたらす可能性がありますと、douncing を起動した後の液体の表面を壊さないように気をつけてください。</li>
	<li>細胞 (すなわち、hela 細胞、HEK293T)、寒さ、1x PBS で 1 回細胞をすすいでください。PBS を外し、セル換散バッファーの最小ボリュームに適用し、細胞スクレーパーと。 注: の上記のセル行細胞 6 ウェル プレート当たり 5 x 10 の5セルでメッキ, 付近の confluency に成長して.1 つはよくマウス線条体に使用されるボリュームと同等です。</li>
	<li>転送磨砕液や細胞ライセートにラベル「不溶性」チューブと氷 1 h (開始クロック最終管が処理された後) に溶解します。 注: セル lysates のカップ培養を開始する前に細胞塊を破るために数回を刻んだ。気泡の形成を避けるため。簡単に渦パルス 1 の各サンプル溶解の途中で s。</li>
	<li>20 分間 15,000 × g で 4 ° C でサンプルを遠心します。</li>
	<li>「水溶性」分数として清を回復します。<ol>
<li>ピペットを使用して削除、これは分数を汚染するとペレット/曇りの層を邪魔しないように注意してください。「水溶性」にピペットで移しなさい可溶性画分は、チューブをラベル付けします。氷の上を保ちます。</li>
	</ol>
</li>
	<li>各洗浄後 5 分 15,000 × g で 4 ° C で 500 μ L の換散バッファー (x2) と遠心分離機でペレットを洗浄します。オフ戻曇り層の一部が場合、それは大丈夫です。</li>
	<li>最終的な洗浄の後組織ペレットだけを残して残りの洗浄バッファーのすべてを削除します。 注意: このポイントから、不溶性サンプルは必要があります常温保管今。</li>
	<li>X μ L 換散バッファー 4 %sds を添加したペレットを再懸濁します (ペレット サイズに応じて音量を調整することができます;テーブル 1を参照)。</li>
	<li>30 の各サンプルを超音波プローブ超音波発生装置で室温で s (125 ワット、20 kHz、パルス、振幅 40%、材料表を参照してください)。</li>
	<li>サンプル (沸騰ローリング) を煮る 30 分;簡単に遠心分離機 (15 s) 任意のサンプルを失わないよう 6,000 x g で。</li>
	<li>DC (互換性のある洗剤) 蛋白質の試金の実行 (材料の表を参照してください) または Lowry 蛋白質の試金 (細胞培養用それぞれの換散バッファーのサンプルの 1/5、1/10 組織溶解液のための開始の希釈を推奨) タンパク質の濃度を測定します。 注: タンパク質の濃度は、高濃縮のための DC またはロウリー アッセイを使用して読み取る必要があります。</li>
	<li>凍結融解の問題を避けるために 50 μ L バッチに割り切れるサンプル。 注: プロトコルを一時停止できるここでさらに生化学分析の前に-20 ° C で両方の分数を格納することによって</li>
</ol>3 西部のしみと定量化
<ol><li>実行報告として西部のしみ他1,13サンプル バッファー (ローディングの染料、削減 x 1 x 1.6) で 1:1 の 30 μ g 希釈します。 注: 分数あたり蛋白質の 30 μ g 十分、しかし最適解像度に調整することができます。3-8% トリス酢酸ポリアクリルアミドゲル不溶性画分を使用してください。4-12% ビス トリス ゲルは可溶性画分に適しています。</li>
	<li>簡単に 5 分遠心分離機のサンプル (沸騰) を沸騰 (15 s) サンプルを収集するために 6,000 x g で。</li>
	<li>減らされたサンプルの製造元のプロトコルに従って SDS-PAGE および西部のしみ、分数を分析します。 注: 不溶性画分を 0.45 μ M 硝酸セルロースの膜に転送されたとき最もよい解決します。0.45 または 0.2 μ M 硝酸セルロースは興味の蛋白質のサイズによって可溶性画分に使用できます。高分子材料の最適な移転に必要ないくつかの最適化があります。</li>
	<li>タンパク質効率の読み込みを視覚化するタンパク質全体染色 (例えばMEMcode、参照テーブルの材料) で膜を染色します。 注: 西部のしみは、平均輝度によって分析できます。可溶性画分は、実験システムに適した家は維持蛋白質の最高に正規化することができます。相対的な蛋白質量として不溶性画分は、可逆的なタンパク質染色または別のしみにヒストン 3 正規化によって確認されました。</li>
</ol>

<ol>
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著者が明らかに何もありません。

定量的な一貫性のある結果を確保するため上記のプロトコルのいくつかの対策が必要です。両方分数で mHTT が複数の凍結融解時に時間をかけて集計を自発的に形成するまず、特に高濃度で。したがって、タンパク質プレップの因数を凍結し、上記のプロトコルで説明されているように分析を実行する前に必要な量のみを解凍するが重要です。さらに、不溶性画分利回り融解時に白い鉛塩法は?...

適切な折りたたみをするタンパク質品質管理ネットワークの中断、細胞蛋白質の低下はアルツハイマー病 (AD)、パーキンソン病 (PD)、ハンチントン病 (HD)、病理学に可能性が高い中央および他の「タンパク」2,3 を障害します。プロテオステイシス ネットワーク コンポーネントおよび病理学への貢献の詳細な理解したがって改善の治療上の介在を開発することが重要です。HD は polyglutamines (polyQ) (HTT) huntingtin 蛋白質4での拡張されたストレッチの結果 HD 遺伝子内の CAG リピートの異常な拡張によって引き起こされます。HD 発症の一貫した病理学的機能、蓄積、および正常細胞の恒常性と機能にいくつかの方法で干渉する示されている HTT 脳の地域および末梢組織での集計は、結果として得られる折りたたみとして、します。5,6。 中の HTT は普遍的表現、線条体中とげだらけニューロンは、選択的に脆弱性が存在と影響を受ける最もあからさま HD 病因にも関連付けられている注目すべき皮質萎縮。
脳内 HD 患者と動物モデルの HTT の集合体の形成は通常病気の進行とドミナント ネガティブ益 mHTT7の関数のプロキシ マーカーを務めています。どのタンパク質の折りたたみと集約に貢献するかもしれない mHTT 誘発毒性正確なメカニズムは不明と様々 な動物モデルの HD 患者の頭脳のこれらの介在物の生成は、不変、必然的な機能です。介在物表示を蓄積した HTT 断片を N 末端の主構成するその分解を示す polyQ を拡大し、9 8,をスプライシングの代替と同様、フルレングスの HTT の処理再生ことがあります、HTT ビジュアルエフェクト N 末端フラグメントの病因に重要な役割は、急速に集計することができます、核と加速や集計プロセス10を伝達する HTT の病理学的フォームを構成するかもしれない。
ただし、これらの介在物の存在では、HTT による毒性や細胞死11と必ずしも関連しません。HTT は不溶性の集計および介在物12に可溶性オリゴマー種と β シート線維を通じて水溶性モノマーから集計プロセスを経るに提案されています。標準生化学的アッセイを介してこれらの様々 な蛋白質種を解決する低洗剤換散バッファーで安定性と標準的な生化学的アッセイを使用して可視化の難しさのためフィールドで課題となっています。したがって、方法論的考察、程度や蓄積・集計 mHTT のパターンを検出するに重要です。
ここで提示されたプロトコルは HTT は、具体的に HD 病因と病気進行1,13 と密接に追跡するために表示される不溶性の HMW HTT 種の形成のいくつかの中間を可視化するメソッドを提供します。、14。解決および mHTT の複数の種を追跡することは、研究者に病因を研究し、変調して病気の発症に影響を与える潜在的な治療上の介在を評価する生化学的なツールを提供します。

<table>
	<tbody>
		<tr>
			<td>Sterile Filter</td>
			<td>Millipore</td>
			<td>SCGP505RE</td>
			<td>Screw cap, sterile vaccum filter</td>
		</tr>
		<tr>
			<td>1 mL Tissue Grinder, Dounce</td>
			<td>Wheaton</td>
			<td>357538</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Model Q125</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Protein Assay</td>
			<td>Biorad</td>
			<td>5000111</td>
			<td>Comparable to Lowry assay</td>
		</tr>
		<tr>
			<td>Tris 1M buffer solution</td>
			<td>Alfa Aesar</td>
			<td>J60636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M solution</td>
			<td>Teknova</td>
			<td>S0251</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS solution</td>
			<td>Teknova</td>
			<td>S0295</td>
			<td></td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Sigma</td>
			<td>E1271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethylsulfony flouride</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>create 100mM stock solution in 100%EtOH, store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>Create 0.5M stock solution in water</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td>Create 10mg/ml stock solution in water</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Sigma</td>
			<td>S4504</td>
			<td>Create 500mM stock solution in water</td>
		</tr>
		<tr>
			<td>Anti-HTT</td>
			<td>Millipore</td>
			<td>MAB5492</td>
			<td>Use 1:1000 for western blot, 1:500 for filter retardation assay</td>
		</tr>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Novus Biologicals</td>
			<td>NB100-56875</td>
			<td>Use 1:1000 for soluble western blot</td>
		</tr>
	</tbody>
</table>

fractionation for resolution of soluble and insoluble huntingtin species

誤って折りたたまれたタンパク質の蓄積は、ハンティントン病 (HD) および他の多くの神経変性疾患の中心です。具体的には、HD の病理学的特徴は高分子量複合体に突然変異体の HTT (mHTT) 蛋白の異常な蓄積、断片および他の蛋白質の細胞内封入体が構成されます。測定および蛍光顕微鏡、西部のしみの分析、フィルターなどの mHTT を含む集計の様々 な形の貢献を理解する従来の方法は、アッセイをトラップします。
ただし、これらのメソッドのほとんどは、特定の構造、したがって mHTT タンパク質フラックス集計溶解度と解像度の複雑な性質のための完全な状態を解決できません。
集計 mHTT の変更されたフォームと複合体の様々 な識別、分離、細胞凝集体とフラグメントの可溶化は必須です。ここで分離し、可溶性 mHTT、モノマー、オリゴマー、フラグメントを視覚化する手法について述べるし、不溶性の高分子量分画法蓄積 mHTT 種。HMW mHTT を追跡、病気の進行とマウス動作の読み出しに対応し、有益ある特定の治療上の介在の1によって調整されています。この方法は、マウスの脳、末梢の組織および細胞培養で使用できるが他のモデル システムや疾患のコンテキストに適応することがあります。

水溶性と不溶性のセル lysates 分別を次の解像度は、ウエスタン解析とフィルター障害アッセイ (図 2) を使用して検出できます。例として HEK293T 細胞のトランスフェクション試薬 (例えば、lipofectamine 2000) を使用してを導入させた、HTT エクソン 1 エンコード cDNA 97 グルタミンを含む繰り返し15ポリ プロリンの豊富な地域に?...

Watch this Scientific Journal Video about Fractionation for Resolution of Soluble and Insoluble Huntingtin Species at JoVE.com

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

不溶解性および溶ける変異ハンチンチン種マウス脳と細胞培養からの分別の方法を説明します。記載されているメソッドは、特性評価および huntingtin 蛋白質フラックスと病気の発症と摂動存在下での蛋白質の恒常性の分析にエイズの定量化の便利

水溶性と不溶性の Huntingtin 種の解像度の分別

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

fractionation-for-resolution-soluble-insoluble-huntingtin

Research

JoVE Journal

Biochemistry

9.5K ビュー.  University of California Irvine. 不溶解性および溶ける変異ハンチンチン種マウス脳と細胞培養からの分別の方法を説明します。記載されているメソッドは、特性評価および huntingtin 蛋白質フラックスと病気の発症と摂動存在下での蛋白質の恒常性の分析にエイズの定量化の便利

ビデオ: Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脳膜分画：<em&gt; Ex Vivo</em&gt; Subsynaptic Protein Localizationを評価するアプローチ

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

生化学

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

人間の死後脳から洗剤不溶性タンパク質凝集体の濃縮

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

高効率巻き戻しと受容体リガンド結合領域の浄化法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch.
The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (&#8805;42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin related modifier (SUMO). The simplicity and efficiency of the strategy will eliminate the need to use non-native sequences of Httex1, thus making this protein more accessible to researchers and improving the reproducibility of experiments across different laboratories. We believe that these advances will also facilitate future studies aimed at elucidating the structure-function relationship of Htt as well as developing novel diagnostic tools and therapies to treat or slow the progression of HD.

Here, we present a robust and optimized protocol for the production of milligram quantities of native, tag-free monomers and fibrils of the exon1 of the Huntingtin protein (Httex1) based on the transient fusion of small ubiquitin related modifier (SUMO).

ネイティブ、タグなしの Huntingtin Exon1 モノマーと相撲の融合戦略を使用して線維の生成

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

フマリアルセトーアセテートヒドロラーゼドメイン含有タンパク質の発現、精製、結晶化、酵素アッセイ

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

一過性発現システムを用いた哺乳類細胞における完全長ヒトハンチンチン変異体の効率的かつスケーラブルな生産

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

豚腎臓およびマウス肝臓からのFAHD1タンパク質の抽出および精製

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.
Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.
The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

マウス組織における安定微小管、不安定な微小管、遊離チューブリンを分離するための定量的微小管分画技術

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

水溶性と不溶性の Huntingtin 種の解像度の分別

要約

さらに動画を探す

この動画の章

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

脳膜分画：

人間の死後脳から洗剤不溶性タンパク質凝集体の濃縮

高効率巻き戻しと受容体リガンド結合領域の浄化法

ネイティブ、タグなしの Huntingtin Exon1 モノマーと相撲の融合戦略を使用して線維の生成

フマリアルセトーアセテートヒドロラーゼドメイン含有タンパク質の発現、精製、結晶化、酵素アッセイ

一過性発現システムを用いた哺乳類細胞における完全長ヒトハンチンチン変異体の効率的かつスケーラブルな生産

豚腎臓およびマウス肝臓からのFAHD1タンパク質の抽出および精製

マウス組織における安定微小管、不安定な微小管、遊離チューブリンを分離するための定量的微小管分画技術

真核細胞からのトランスレーショナル複合体の迅速なインビボ固定と分離

水溶性と不溶性の Huntingtin 種の解像度の分別

要約

さらに動画を探す

この動画の章

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

脳膜分画：

人間の死後脳から洗剤不溶性タンパク質凝集体の濃縮

高効率巻き戻しと受容体リガンド結合領域の浄化法

ネイティブ、タグなしの Huntingtin Exon1 モノマーと相撲の融合戦略を使用して線維の生成

フマリアルセトーアセテートヒドロラーゼドメイン含有タンパク質の発現、精製、結晶化、酵素アッセイ

一過性発現システムを用いた哺乳類細胞における完全長ヒトハンチンチン変異体の効率的かつスケーラブルな生産

豚腎臓およびマウス肝臓からのFAHD1タンパク質の抽出および精製

マウス組織における安定微小管、不安定な微小管、遊離チューブリンを分離するための定量的微小管分画技術

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

真核細胞からのトランスレーショナル複合体の迅速なインビボ固定と分離