このプロジェクトは、立ち上げ資金のためのロチェスターの大学、GM127652、NIH 助成金 EY025290 で賄われていた。

1. 生産 BacMam 式バキュロ
<ol><li>BacMam ベクトル18 GFP 10 続くタバコ Etch ウイルス (TEV) プロテアーゼ認識シーケンス、ペグに必要な哺乳類 bestrophin 蛋白質のコーディング シーケンスを挿入、タンパク質の C 末端タグ x。</li>
	<li>一過性発現プラスミドを接着剤 HEK293 細胞19,20,21,22,23に transfect します。10 倍や 20 倍の倍率、488 nm 励起ソース 510 nm 発光フィルター (図 1 a) と蛍光顕微鏡下で GFP 融合タンパク質の発現を確認してください。 注: ポリマー ベースのトランスフェクションには、1 μ g のプラスミド DNA HEK293 細胞の 35 mm ディッシュ用、日常的に実行されます。</li>
	<li>前述の16,17として Sf9 昆虫細胞におけるバキュロを生成します。 注: 通路 3 (P3) ウイルス、タンパク質生産のため F HEK293 細胞に感染するために使用が、4 ° C で保存できる 1 ヶ月。</li>
	<li>プラーク形成アッセイ16,17P3 ウイルスの力価 (感染単位) を決定します。 注: 興味の蛋白質に GFP タグは、感染ユニットを確認するより高速な方法はウイルスのシリアル希薄と Sf9 細胞に感染して 48 時間後蛍光セルの数をカウントします。</li>
</ol>2. タンパク質発現
<ol><li>8% の CO2と 37 ° C 加湿インキュベーター HEK293 F 式培地と浮遊培養で HEK293 F 細胞を維持します。使い捨て文化フラスコ 2 〜 2.5 倍より大きい文化のボリュームよりも、回転で 135 回転軌道プラットフォーム上のカルチャを使用します。 注: お勧め 5 と 30 の通路の間にあるとき、細胞に感染して細胞文化フードの下でこれらのアクションを実行します。</li>
	<li>ウイルス感染前に 24 h は細胞培養の 15 μ 因数にトリパン ブルー色素の 15 μ L を追加してセル密度と生存率の顕微鏡下での検定を使用しています。各媒体の 500 ml で 37 ° C に加温 2 x 2 L フラスコに 10 の6セル/mL x 0.6 に細胞を希釈します。 注: 死んだ細胞がブルーで染色トリパン ブルー.目的のセル実行可能性は、> 95%。</li>
	<li>感染の日、細胞密度と生存率 (手順 2.2) を確認します。 注: の高い細胞生存率 > 95% は効率的な感染および蛋白質の表現のために不可欠です。予想される細胞密度は、~1.0 x 106セル/mL (10 6/mL の細胞培養 x 0.6 の 2 x 500 mL から成長した一晩) です。予想される総細胞数は ~1.0 x 109です。</li>
	<li>5 の感染 (MOI) の多様性で細胞に P3 バキュロを追加します。接種するウイルスの量を調べるには、するには、次の式を使用します。 <img alt="Equation" src="/files/ftp_upload/57832/57832eq1.jpg">
</li>
	<li>24 h の 8% の CO2と加湿の 37 ° C のインキュベーターで 135 の rpm の軌道プラットフォーム上のセルを振る。</li>
	<li>細胞培養に酪酸の 10 mM を追加し、48 時間の培養を続けます。 注: いくつかの蛋白質の細胞培養温度を 30 ° C に削減して後ナトリウム酪酸添加が式と安定性を向上します。</li>
	<li>10 倍や 20 倍の倍率、488 nm 励起ソース、510 nm 発光フィルター蛍光顕微鏡、割合と直接 (図 1 b) 蛋白質 (bestrophin-GFP-10xHis) 収量を表す緑の細胞の明るさを確認します。 注: 緑の細胞の割合によって計算されます: (緑色のセル/合計セル) × 100。良いタンパク質発現レベルは、顕微鏡下で強力な蛍光グリーンで示されます。</li>
	<li>4 ° C、20 分削除上清に 1,000 × g で遠心分離によって細胞を収穫し、リン酸緩衝生理食塩水 (PBS) ~ 80 mL の最終巻に、細胞ペレットを再中断します。分割 2 x 50 mL の円錐管に 4 ° C で 1,000 x g で 20 分間遠心細胞懸濁液は-80 ° C で細胞ペレットを格納します。</li>
</ol>3. 蛋白質の浄化
<ol><li>解凍するように 10-15 分間室温で攪拌水浴の細胞ペレットを含む円錐管を孵化させなさい。0.1 〜 1.0 プロテイナーゼ阻害剤 (特異ペプスタチン A、Leupeptin アプロチニン フッ化) 補われる (w/v) 量のバッファー (表 1) (例えば、 20 mL のバッファーで細胞ペレットの 10 g) x 2 のセル中断再 mM。上下ピペット広範囲に均一な細胞の懸濁液を得る。</li>
	<li>高圧ホモジナイザーを用いた細胞を溶解させます。細胞懸濁液を通すホモジナイザーの合計のための 7-10 MPa で 3-4 回完全な均質化を達成するために。ラウンドの間 2-3 分のため氷の上細胞ライセートをしてください。</li>
	<li>細胞ライセートに洗剤 [例えば2 w/v ソル グレード n-ドデシル-β-D-Maltopyranoside (DDM)] を追加します。膜タンパク質を抽出する 20 の ° C で 1 時間攪拌で孵化させなさい。 注: タイプの洗剤は、各蛋白質ターゲット18,24用に最適化する必要があります。</li>
	<li>1 h の 4 ° C で遠心で 150,000 × g で遠心分離機します。 注: 次の手順のすべては 4 ° C で実行されます。</li>
	<li>明確な細胞ライセートと流れ、5 mL の彼のトラップ Ni2 +-国税庁アフィニ ティー ・ カラムを通してそれは事前 (表 1) のバッファーで平衡を収集します。 注: 遠心分離の後明確な細胞ライセートは下部にペレットと曇りの層の上にサンドイッチされ。10 mL のピペットを使用して、慎重にクリア ライセートを収集、ライセートの数ミリリットル最後の 1 mL ピペットに切り替えています。Ni2 +列を詰まらせることが、ペレットを避けるためただし、最上位のレイヤーの少量は大丈夫ですまたは 1.5 μ m フィルターで除外することができます。</li>
	<li>洗浄バッファー B 25 mL とし、40 mL のバッファー C 列 (表 2 および 3、それぞれ)。 注: これは休憩合計浄化は最大 12 時間をかかることがあり、タンパク質は一晩の列に接続されている間安定している残ることができる良い場所です。</li>
	<li>タンパク質の高速液体クロマトグラフィー (FPLC) システムに列を添付し、13 mL のバッファー D を持つ列から (表 3) をもつタンパク質分画を溶出します。UV 吸光度読み出しによるとタンパク質濃縮分数を収集します。 注: 、FPLC 条件は次のとおり: 1.0 mL 分数サイズ、0.3 MPa、流量を 0.5 mL/min に設定前のカラムの圧力警報。</li>
	<li>微量分光光度計で溶離されたタンパク質製品の濃度を測定します。280 吸光度を読み nm。</li>
	<li>(省略可能)10xHis GFP タグを削除するには、1:1 の質量比で TEV プロテアーゼを追加し、4 の ° C で 30 分間インキュベートします。 メモ: タグ、関数または浄化されたチャネルの構造には影響があります。TEV ボリューム ステップ 3.7 3.8 から収集された蛋白質の濃度から算出します。例えば、10 mL の溶出物を収集し、0.1 mg/ml、総蛋白測定質量は TEV の 10 × 0.1 = 1 mg、および 1 mg が消化に使用されます。</li>
	<li>(50 または 100 kDa) 15 ml 遠心蛋白質を集中 (2 mL サンプル読み込みループ FPLC) の 400-500 μ L の最終巻に 4 ° C で 4,000 x g で回してフィルター ユニット。 注: 濃度の遠心時間は濃度と蛋白質のサイズによって異なります。過集中を避けるため、可能性があります蛋白質の沈殿物を引き起こす、最初は、10 分間スピンし、残りのボリュームを観察および後続のスピンに時間を見積もる (例えば2-5 分) というように、最終的なボリュームを取得します。フィルターの下に引かれているタンパク質を分散させるためにスピン間ピペットで移しなさい。</li>
	<li>新しい 1.5 mL チューブまたは削除する任意の沈殿物の泡集中製品から高濃度のタンパク質を転送します。スピン > 4 ° C および収集新しい 1.5 mL チューブに上清を 5-10 分のための 12,000 × g。</li>
	<li>あらかじめバッファー E (表 5) が平衡サイズ排除カラム 1 mL シリンジとサイズ排除クロマトグラフィー用 FPLC システムのラウンド チップ針で最終製品をロードします。 注: 、FPLC 条件は次のとおり: 0.5 mL 分数サイズ、2.50 MPa に設定前のカラムの圧力警報、30 mL のバッファー E に設定流量(表 5)、流量を 0.5 mL/min に設定します。</li>
	<li>行儀蛋白質 (図 2 a) 単一のピークとして実行 (図 2 a) をピークに対応するタンパク質 fraction(s) を集めることに注意します。</li>
	<li>(ステップ 3.10 のように同じ分子量カットオフ) の 4 mL または 0.5 mL の遠心フィルター ユニットとスピンを最終濃度 5-10 μ g/μ L の 4 ° C で 4,000 x g でタンパク質を集中します。分光光度計を使用して、最終製品濃度 (ステップ 3.8) をチェックします。 注: 遠心時間、最終製品ボリュームことができます浄化試験間で異なるとは異なります。遠心分離は、通常、10 〜 20 分かかります。フィルターの下に引かれているタンパク質を分散させるためにスピン間のピペットにそれをお勧めします。</li>
	<li>高濃度のタンパク質を新しい 1.5 mL チューブに転送します。遠心分離によって任意の沈殿物を削除 > 4 ° C で 5 ~ 10 分の 12,000 × g上清を新しい 1.5 mL チューブに転送します。ストレージのための 10 の μ 因数-80 ° c、9 V/cm (図 2 b) 4 15% 勾配 SDS ページのゲル上で実行する 1 つの 2-5 μ L 小さい因数を保存します。</li>
	<li>質量8によって浄化された蛋白質のアイデンティティを確認します。 注: 関数のIn vitro解析と浄化された蛋白質の構造平面脂質二分子膜と x 線結晶構造解析、それぞれ5,8を介して実行できます。</li>
</ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+RGK+GTPases+differentially+use+alpha1-+and+auxiliary+beta-binding-dependent+mechanisms+to+inhibit+CaV1.2/CaV2.2+channels.">Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.</a> Public Library of Science One. 7 (5), e37079 (2012).</li><li>Yang, T., Suhail, Y., Dalton, S., Kernan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+molecules+for+inducibly+inactivating+CaV+channels.">Genetically encoded molecules for inducibly inactivating CaV channels.</a> Nature Chemical Biology. 3 (12), 795-804 (2007).</li><li>Yang, T., Xu, X., Kernan, T., Wu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> Journal of Physiology. 588 (Pt 10), 1665-1681 (2010).</li><li>Kawate, T., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-detection+size-exclusion+chromatography+for+precrystallization+screening+of+integral+membrane+proteins.">Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.</a> Structure. 14 (4), 673-681 (2006).</li><li>Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+cryo-electron+microscopy+(cryo-EM)+and+cross-linking+mass+spectrometry+(CX-MS)+for+structural+elucidation+of+large+protein+assemblies.">Combining cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (CX-MS) for structural elucidation of large protein assemblies.</a> Currents Opinions in Structural Biology. 46, 157-168 (2017).</li><li>Sun, W., Zheng, W., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+and+development+for+rare+genetic+disorders.">Drug discovery and development for rare genetic disorders.</a> American Journal of Medical Genetics Part A. 173 (9), 2307-2322 (2017).</li></ol>

著者が明らかに何もありません。

このプロトコルでは、将来の in vitro解析に使用される哺乳類 bestrophin イオン チャネルの発現と精製に有用なパイプラインについて説明します。サイズ排除クロマトグラフィー用 FPLC デバイス、シリンジ ポンプはアフィニ ティー ・ クロマトグラフィーのバインド、洗濯、溶出性などのすべてのステップのために十分。列を (注射器) でプッシュ ソリューションにシリンジ ポンプを使?...

Bestrophins は、人間1細菌からさまざまな種を保存するイオン チャネルの家族です。ヒトでは、 BEST1の遺伝子、染色体 11q12.3 に位置するエンコード膜タンパク質 Bestrophin-1 (BEST1) 目2,3 の網膜色素上皮 (RPE) 細胞の基底膜で主に表される ,4。うち最初の 〜 350 種間保存性が高いと、膜貫通領域を含む、585 アミノ酸から成る BEST1 人間1,5,6に CaCC を果たします。さらに、鶏の BEST1 同族体および肺炎桿菌の両方として機能する homopentamers7、8、進化を通じて保全の高レベルを示唆しています。
ヒトでは、200 以上BEST1遺伝子変異を臨床的に bestrophinopathies1,9と呼ばれる網膜変性疾患のグループにリンクされています。ベスト病、成人発症卵黄状黄斑ジストロフィー、常染色体優性の支配的な vitreoretinochoroidopathy、常染色体優性劣性 bestrophinopathy、網膜色素変性症3,4 など 5 つの特定の bestrophinopathies が報告されています。 ,,1011,12,13,14。視力の低下、さらには失明につながるが、これらの病気、現在治療されます。治療上の処置および可能性のある個人化された薬を開発するために関数と BEST1 チャネル15の構造のこれらのBEST1病気を引き起こす突然変異の影響を理解するが重要です。これらの目的のための研究者の in vitro解析5,8を行い精製 bestrophin (野生型および変異) チャンネルを取得する必要があります。
最初の重要なステップは、哺乳類細胞における高い種から bestrophin チャンネルの式です。このプロトコルが堅牢な発現の最適化された BacMam ベクトル (BacMam pEG) を利用して F HEK293 細胞 (BacMam システム) のバキュロ ウイルス伝達はクローン膜タンパク質16,17を表現する強力な方法は、ターゲット蛋白質18, この場合 bestrophin 哺乳類ホモログであります。このベクターは、G タンパク質共役受容体、核内受容体、イオン チャンネル18など、各種の膜タンパク質の発現のために使用されています。また、作り出されたタンパク質が結晶18に適している証拠です。F HEK293 細胞における発現の高レベルの蛋白質できます、精製するクロマトグラフィー具体的には、bestrophins、場合親和性とサイズ排除クロマトグラフィーを使用することができます。
このプロトコルは bestrophin チャンネルの微調整が、浄化された蛋白質は関数と平面脂質二分子膜とそれぞれ5,8、x 線結晶構造解析による構造を分析できます。完全に、これらのテクニックは、bestrophins と他のイオン チャネルの構造と機能の調査のため強力なパイプラインを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">AC327265000&#160;</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC446212500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">G33-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imidazole</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC301870010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgCl2</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC197530010&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AA4058704&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aprotinin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ63039MA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ61188MB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin A</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ20037MB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC215740050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM sol-grade</td>
			<td style="width:148px;">Anatrace</td>
			<td style="width:119px;">D310S</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM anagrade</td>
			<td style="width:148px;">Anatrace</td>
			<td style="width:119px;">D310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf-900 II SFM</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:119px;">10902179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeStyle medium</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">12338018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop spectrophotometer</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High pressure homogenizer</td>
			<td style="width:148px;">Avestin</td>
			<td style="width:119px;">Emulsiflex-C5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HisTrap column</td>
			<td style="width:148px;">GE</td>
			<td>17-5248-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex-200 column</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">28990944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKTA Pure</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">29018224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-15 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-4 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC810024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-0.5 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC505024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optima XE-90 Ultracentrifuge</td>
			<td style="width:148px;">Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN precast gel</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>4561084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PolyJet transfection reagent</td>
			<td style="width:148px;">SignaGen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEG BacMam vector</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td>Obtained from the Gouaux lab at Vollum Institute</td>
		</tr>
	</tbody>
</table>

expression and purification of mammalian bestrophin ion channels

人間のゲノムは、4 つの bestrophin paralogs、すなわち BEST1、BEST2、ベスト 3、ベスト 4 をエンコードします。BEST1の遺伝子によって符号化される BEST1 は、Ca2 +-アクティブに Cl-チャネル (CaCC) 主に網膜色素上皮 (RPE) で表されます。BEST1 の生理学的および病理学的意義がBEST1遺伝子の 200 以上の異なる変異が最高の卵黄など少なくとも 5 つの網膜変性疾患のスペクトルに遺伝的リンクされているという事実によって強調表示されます。黄斑ジストロフィー (疾患)。したがって、非常に大きな意義を保持 bestrophin チャネルを単一分子レベルでの生物物理を理解すること。ただし、精製された哺乳類のイオン チャネルを得ることはやりがいのある仕事ではしばしばです。ここで、哺乳類 bestrophin BacMam バキュロ ウイルスの遺伝子の転送システムと親和性とサイズ排除クロマトグラフィーの浄化蛋白質の表現のためのプロトコルを報告します。浄化された蛋白質脂質二重層と結晶の電気生理学的記録など、以降の機能・構造解析に活用される可能性があります。重要なは、このパイプラインは他のイオン チャネルの構造と機能を研究する合わせることができます。

一過性トランスフェクション接着 HEK293 細胞 (図 1 a) の蛍光強度は、サスペンション F HEK293 細胞 (図 1 b) に投影されたタンパク質発現レベルの良い指標です。表現の構成要素を変更することを検討する場合はターゲット蛋白質が発現された、または、誤 HEK293 細胞にトランスフェクション後ローカライズされて、お勧め (e...

Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

Expression and Purification of Mammalian Bestrophin Ion Channels

哺乳類 Bestrophin イオン チャネルの発現と精製

イオン チャネルの浄化はしばしば困難、しかし、一度は、それが悪意のチャネルの構造と機能の生体外で調査。ここでは、式と哺乳類 bestrophin 蛋白質、Ca2 +の家族の浄化の段階的な手順を述べる-Cl-チャンネルを活性化します。

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

expression-and-purification-of-mammalian-bestrophin-ion-channels

Research

JoVE Journal

Biochemistry

8.3K ビュー.  University of Rochester, School of Medicine and Dentistry. イオン チャネルの浄化はしばしば困難、しかし、一度は、それが悪意のチャネルの構造と機能の生体外で調査。ここでは、式と哺乳類 bestrophin 蛋白質、Ca2 +の家族の浄化の段階的な手順を述べる-Cl-チャンネルを活性化します。

ビデオ: Expression and Purification of Mammalian Bestrophin Ion Channels

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

にバインドされた熱安定、発現、精製、およびヒトセロトニントランスポーターの結晶化<em&gt; S</em&gt; - シタロプラム

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

生化学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12の発現、精製および抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

オープン パッチ ・ クランプのピペットで非撹拌の境界層におけるイオン濃度の測定: 流体によるイオン チャネルの制御の流れ

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

式と単一粒子の低温電子顕微鏡による構造決定のための人間脂質感受性陽イオン チャネル TRPC3 の精製

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

式、可溶化、および真核生物ホウ酸輸送体の浄化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

ヌクレアーゼフリー酸素スカベンジャープロトカテク酸プロトカテクエート3,4-ジオキシゲナーゼの発現と精製

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

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 ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳類組織の富化とコレステロールを伴うゼノプス卵母細胞

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

PCR変異生成、クローニング、発現、高速タンパク質精製プロトコルおよびトリプトファン合成酵素の野生型および変異型の結晶化

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

ガングリオシドの抽出、精製、プロファイリング

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

哺乳類 Bestrophin イオンチャネルの発現と精製

要約

さらに動画を探す

この動画の章

にバインドされた熱安定、発現、精製、およびヒトセロトニントランスポーターの結晶化

S100A12の発現、精製および抗菌活性

オープンパッチ・クランプのピペットで非撹拌の境界層におけるイオン濃度の測定: 流体によるイオンチャネルの制御の流れ

式と単一粒子の低温電子顕微鏡による構造決定のための人間脂質感受性陽イオンチャネル TRPC3 の精製

式、可溶化、および真核生物ホウ酸輸送体の浄化

ヌクレアーゼフリー酸素スカベンジャープロトカテク酸プロトカテクエート3,4-ジオキシゲナーゼの発現と精製

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

哺乳類組織の富化とコレステロールを伴うゼノプス卵母細胞

PCR変異生成、クローニング、発現、高速タンパク質精製プロトコルおよびトリプトファン合成酵素の野生型および変異型の結晶化

ガングリオシドの抽出、精製、プロファイリング

哺乳類 Bestrophin イオン チャネルの発現と精製

要約

さらに動画を探す

この動画の章

にバインドされた熱安定、発現、精製、およびヒトセロトニントランスポーターの結晶化

S100A12の発現、精製および抗菌活性

オープン パッチ ・ クランプのピペットで非撹拌の境界層におけるイオン濃度の測定: 流体によるイオン チャネルの制御の流れ

式と単一粒子の低温電子顕微鏡による構造決定のための人間脂質感受性陽イオン チャネル TRPC3 の精製

式、可溶化、および真核生物ホウ酸輸送体の浄化

ヌクレアーゼフリー酸素スカベンジャープロトカテク酸プロトカテクエート3,4-ジオキシゲナーゼの発現と精製

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

哺乳類組織の富化とコレステロールを伴うゼノプス卵母細胞

PCR変異生成、クローニング、発現、高速タンパク質精製プロトコルおよびトリプトファン合成酵素の野生型および変異型の結晶化

ガングリオシドの抽出、精製、プロファイリング

哺乳類 Bestrophin イオンチャネルの発現と精製

オープンパッチ・クランプのピペットで非撹拌の境界層におけるイオン濃度の測定: 流体によるイオンチャネルの制御の流れ

式と単一粒子の低温電子顕微鏡による構造決定のための人間脂質感受性陽イオンチャネル TRPC3 の精製