このプロジェクトは、全米科学財団と生体分子科学学会からCJJへの補助金によって支えられている。

1。 1日目： 緑膿菌からMexBはpFB101によってエンコードされます。 MexB遺伝子は、P.から増幅された緑膿菌ゲノムDNAおよびpET30b +ベクトルのNdeI及びXhoI制限部位に挿入。構造はC末端ヘキサヒスチジンタグが含まれています。 <ol><li>プラスミドを大腸菌を形質転換するのに使用され大腸菌は、C43（DE3）12株、および形質転換体を30 ug / mlとカナマイシンを含むLB寒天上にプレーティングされています。 </li></ol> 2。 2日目：一晩培養： <ol start="2"><li>夕方には、30 ug / mlとカナマイシンを含む4 X 3 mlのLB培養液は、新鮮な形質転換体のコロニーから接種されています。また、培養は、凍結パーマから接種することができます。 これらの小さな文化は37℃で一晩ローラー上に成長されています。 </li></ol> 3。 3日目：6リットルの文化を育てる。 <ol start="3"><li>午前中は、30 ug / mlとカナマイシンを含む150mLのLBに接種するのに一晩培養を使用してください。 37シェーカー上で° Cで培養して成長する。 </li><li>午後には、フェルンバッハのフラスコで30 ug / mlとカナマイシンを含む6 × 1L 2XYTメディアを接種する小さな文化を使う。 （1:40希釈用培養液あたり25mlを使用してください）​​。 37の文化を育て° Cを彼らが0.4から0.6のOD 600に到達するまで、1.5時間程度</li><li>培養物は、適​​切な密度に達すると、0.5mLの1M IPTGを添加することによりタンパク質の発現を誘導する。シェーカーに戻ってすべてのフラスコに入れ、℃で一晩30℃、それらを成長を続けている。 </li></ol> 4。 4日目：細胞を採取し、タンパク質を精製する。 <ol start="6"><li>次のように、緩衝液にプロテアーゼ阻害剤、DNアーゼ、およびリゾチームを追加する：細胞の再懸濁バッファー50mlに、10mgのDNaseI（0.1 mg / mLの最終濃度）、リゾチームの1コンプリートEDTA -フリープロテアーゼ阻害剤のタブレット、とピンチを追加します。 。膜の再懸濁バッファー60 mLに、1プロテアーゼ阻害剤のタブレットを追加する。膜の再懸濁バッファーの別の50 mLに、1プロテアーゼ阻害剤のタブレットを追加する。氷の上ですべての3つのソリューションをしてください。 </li><li>細胞を回収するために大規模な遠心分離機の文化30分5,000 rpmで遠心する。 </li><li> 100 mLの細胞を再懸濁バッファーに懸濁し（50 mMのNAPは、pH 7.0、300mMのNaCl、2mMのMgCl 2、1コンプリートEDTA -フリープロテアーゼ阻害剤のタブレット、リゾチームのは0.1 mg / mlのDNase I、ピンチ） </li><li> 12000 PSI（762ゲージ圧）でフレンチ圧力セルを2回細胞溶液を渡します。瓶の中に細胞ライセートを収集するには、氷の上に冷たいまま。 </li><li> SS34遠心チューブに細胞溶解液を移し、4℃、10,000 rpmで30分間細胞破片を除去するため遠心° CのSS - 34ローターで。 </li><li>慎重にTi647.5超遠心チューブに上清を取り除く。 4Cで40,000 rpmで50分間遠心操作する。上清を捨てる。 </li><li>約で、細胞膜を含むペレットを、懸濁します。膜の再懸濁バッファーを25mL（50 mMのNAPは、pH 7.0、300mMのNaCl、5％グリセロール、1コンプリートEDTA -フリープロテアーゼ阻害剤の錠剤）。 </li><li> 4℃で50分間Ti647.5ローターで40,000 rpmで遠心チューブと遠心分離機℃に膜懸濁液を移す</li><li>上清を捨て、25mLの膜の再懸濁バッファー（50mMのNAPは、pH 7.0、300mMのNaCl、5％グリセロール、1コンプリートEDTA -フリープロテアーゼ阻害剤の錠剤）で洗浄した膜ペレットを再懸濁します。 </li></ol> 5。 TMタンパク質Solublization： <ol start="15"><li>再懸濁した膜（25mLを約）に、6 mLの10％DDM（最後の界面活性剤濃度= 2％DDM）ロック4で混合℃で2時間。を追加する</li><li> 4℃で40分間40,000 rpmで混合物を遠心° C不溶性の蛋白質から可溶性タンパク質の界面活性剤複合体を分離するTi647.5ローターインチMexBタンパク質の洗剤の複合体を含む上清を、保存します。 </li></ol> 6。 IMAC： <ol start="17"><li>再懸濁バッファーで平衡化した爪の金属アフィニティービーズで高速回転から得られた上清を混ぜる。 4でローラーで1時間インキュベート℃に</li><li>重力流カラム本体にスラリーを注ぎ、フロースルーを捨てる。 </li><li> IMAC結合および洗浄バッファー（50 mMリンNAP、pH7で、300mMのNaCl、5％グリセロール、0.2％DDM）を20mL（10カラム体積）でカラムを洗浄</li><li> IMAC溶出バッファー（50mMのNAPは、pH 7.0、300mMのNaCl、5％グリセロール、250 mMイミダゾール、0.2％DDM）でタンパク質を溶出する。 </li><li>溶出画分15μLのサンプルを取る、ポリアクリルアミドゲル電気泳動による分析のために15μL2X SDSサンプルバッファーでそれぞれを混ぜる。微量遠心機で30秒を回転させる。各分画でMexBの量と純度を推定するために10％ポリアクリルアミドSDSゲル上でサンプルを分析する。 </li><li> MexBタンパク質の洗剤の複合体と4でスピンコンセントレータでそれらを集中を含む画分をプール℃のタンパク質がこのsで沈殿しないように注意してくださいTEP。 </li></ol> 7。ゲル濾過カラム： <ol start="23"><li> 24 mlのランニングバッファー（50 mMのNAPは、pH 7.0、300mMのNaCl、5％グリセロール、0.2％β-オクチルグルコシド）とスーパーローズ12 HL 10分の30の列をあらかじめ平衡化し、フラットなベースラインを待ちます。 </li><li>バッファを実行するとループをロードするAKTAシステムをすすぎます。 </li><li>列に適用する前に、シリンジフィルターを用いてタンパク質溶液をフィルタ。 </li><li> 5mgの/ mLまでのタンパク質濃度で、カラムにタンパク質溶液240μLまで原稿をセット。 </li><li>バッファの1.5カラム体積（36 mL）を実行する、0.25 mLの画分を集めた。溶出量を15mL - MexBタンパク質の洗剤の複合体は、約10のピークとして溶出してください。 </li><li>ピーク画分の5μLのサンプルを取る。 2X SDSサンプルバッファーで各サンプル1:1に混ぜる。各分画でMexBの量と純度を推定するために10％ポリアクリルアミドゲル上でサンプルを分析する</li><li>純粋MexBを含む画分プール。 </li></ol> 8。代表的な結果： 図1は、ゲル濾過カラムからのIMACカラムと個々の画分からプールされたカラムの分画とポリアクリルアミドゲルを含んでいる。ゲル濾過カラムの後にタンパク質はクマシー染色したポリアクリルアミドゲルで純粋に表示されます。図2は、カラムからのタンパク質の洗剤複雑な溶出の主要なピークを示すゲル濾過カラムからのトレースが含まれます。 MexBタンパク質の平均収量は2XYT文化6リットルあたり約2ミリグラムです。 <img src="/files/ftp_upload/2134/2134fig1.jpg" alt="図1" /> 図1。 MexBのPDCの精製の​​SDS - PAGEゲル。レーン1、分子量マーカー。 2、IMACの画分をプールされた。 3-7、ゲルろ過カラムの分画。 <img src="/files/ftp_upload/2134/2134fig2.jpg" alt="図2" /> 図2。 MexBタンパク質の洗剤の複合体（PDC）用のゲルろ過の結果の例。

<ol>
	<li>Eda, S., Maseda, H., Nakae, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elegant+means+of+self-protection+in+gram-negative+bacteria+by+recognizing+and+extruding+xenobiotics+from+the+periplasmic+space.">An elegant means of self-protection in gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space.</a> J. Biol. Chem. 278, 2085-2088 (2003).</li><li>Li, X. Z., Ma, D., Livermore, D. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+efflux+pump(s)+in+intrinsic+resistance+of+Pseudomonas+aeruginosa:+active+efflux+as+a+contributing+factor+to+beta-lactam+resistance.">Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance.</a> Antimicrob. 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Agents Chemother. 44, 3322-3327 (2000).</li><li>Okusu, H., Ma, D., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AcrAB+efflux+pump+plays+a+major+role+in+the+antibiotic+resistance+phenotype+of+Escherichia+coli+multiple-antibiotic-resistance+(Mar)+mutants.">AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants.</a> J. Bacteriol. 178, 306-308 (1996).</li><li>Srikumar, R., Kon, T., Gotoh, N., Poole, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Pseudomonas+aeruginosa+multidrug+efflux+pumps+MexA-MexB-OprM+and+MexC-MexD-OprJ+in+a+multidrug-sensitive+Escherichia+coli+strain.">Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD-OprJ in a multidrug-sensitive Escherichia coli strain.</a> Antimicrob. Agents Chemother. 42, 65-71 (1998).</li><li>Tikhonova, E. B., Zgurskaya, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AcrA,+AcrB,+and+TolC+of+Escherichia+coli+Form+a+Stable+Intermembrane+Multidrug+Efflux.">AcrA, AcrB, and TolC of Escherichia coli Form a Stable Intermembrane Multidrug Efflux.</a> Complex. J. Biol. Chem. 279, 32116-3224 (2004).</li><li>Yoneyama, H., Ocakatan, A., Tsuda, M., Nakae, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mex-gene+products+in+antibiotic+extrusion+in+Pseudomonas+aeruginosa.">The role of mex-gene products in antibiotic extrusion in Pseudomonas aeruginosa.</a> Biochem. Biophys. Res. Commun. 233, 611-618 (1997).</li><li>Berger, B. W., Gendron, C. M., Robinson, C. R., Kaler, E. W., Lenhoff, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+protein+and+surfactant+interactions+in+membrane-protein+crystallization.">The role of protein and surfactant interactions in membrane-protein crystallization.</a> Acta. Crystallogr. D Biol. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-production+of+proteins+in+Escherichia+coli:+mutant+hosts+that+allow+synthesis+of+some+membrane+proteins+and+globular+proteins+at+high+levels.">Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels.</a> J. Mol. Biol. 260, 289-298 (1996).</li></ol>

利害の衝突は宣言されません。

多剤耐性に加えて、イオン輸送、細胞間コミュニケーション、小胞輸送、細胞構造の維持、および宿主 - 病原体相互作用を含む多くの重要な細胞活動は、細胞膜に埋め込まれているタンパク質を含む。膜貫通型タンパク質は、既知のタンパク質の30％以上を構成し、今日使用されている医薬品の大半の目標です。膜貫通型タンパク質の不適切な折りたたみや活動は、嚢胞性線維症や糖尿病な?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SDS sample buffer</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>161-0737</td>
<td>&nbsp;</td>
<tr>
<td>C43(DE3) E. coli strain</td>
<td>&nbsp;</td>
<td>Lucigen</td>
<td>60345-1</td>
<td>&nbsp;</td>
<tr>
<td>kanamycin sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K4378</td>
<td>&nbsp;</td>
<tr>
<td>2XYT media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP2466-2</td>
<td>&nbsp;</td>
<tr>
<td>LB media</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC61189-5000</td>
<td>&nbsp;</td>
<tr>
<td>IPTG</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I6758</td>
<td>&nbsp;</td>
<tr>
<td>DNaseI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP3226-1</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L7651</td>
<td>&nbsp;</td>
<tr>
<td>Complete EDTA-free protease inhibitor tablets</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11 873 580 001</td>
<td>&nbsp;</td>
<tr>
<td>NaP monobasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6566</td>
<td>&nbsp;</td>
<tr>
<td>NaP dibasic</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M1028</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-1</td>
<td>&nbsp;</td>
<tr>
<td>n-dodecyl-&beta;-D-maltopyranoside</td>
<td>&nbsp;</td>
<td>Anatrace</td>
<td>D310</td>
<td>&nbsp;</td>
<tr>
<td>15ml tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>See-Saw Rocker</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SSL 4</td>
<td>&nbsp;</td>
<tr>
<td>Talon metal affinity resin</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>635503</td>
<td>&nbsp;</td>
<tr>
<td>imidazole</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>I5513</td>
<td>&nbsp;</td>
<tr>
<td>10% polyacrylamide SDS PAGE gels</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-1454</td>
<td>&nbsp;</td>
<tr>
<td>Tris/glycine/SDS PAGE running buffer</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0732</td>
<td>&nbsp;</td>
<tr>
<td>Kaleidascope prestained molecular weight markers</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0324</td>
<td>&nbsp;</td>
<tr>
<td>Superose 12 30/10 column</td>
<td>&nbsp;</td>
<td>GEHealthcareSuperose</td>
<td>12 10/300 GL</td>
<td>&nbsp;</td>
<tr>
<td>Amicon centrifugal concentrator</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801024</td>
<td>&nbsp;</td>
<tr>
<td>Syringe filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SLFG R04 NL</td>
<td>&nbsp;</td>
<tr>
<td>Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-552-39</td>
<td>&nbsp;</td>
<tr>
<td>Shaker to hold Fernbach flasks</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Akta system</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>J6 Large scale centrifuge with JLA-8.1000 rotor</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 l centrifuge bottles</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>969329</td>
<td>&nbsp;</td>
<tr>
<td>RC-5 centrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SS34 fixed-angle rotor and tubes</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sorvall floor model Ultracentrifuge</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T647.5 rotor and tubes with caps</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>08322</td>
<td>&nbsp;</td>
<tr>
<td>French Pressure Cell</td>
<td>&nbsp;</td>
<td>ThermoScientific</td>
<td>FA-032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

expression, detergent solubilization, and purification of a membrane transporter, the mexb multidrug resistance protein

多剤耐性（MDR）、構造的および機能的に関係のない抗がん剤や抗生物質の広い範囲に耐性であることが癌細胞や病原体の能力は、公衆衛生の現在の深刻な問題です。この多剤耐性がエネルギー依存性の薬剤排出ポンプによるところが大きい。ポンプは、毒性閾値以下にそれらの細胞内濃度を下げる、外部媒体への抗がん剤や抗生物質を排出する。我々は、 緑膿菌 、例えば、怪我や病気の多くのタイプの患者に感染症を引き起こす日和見細菌性病原体、火傷や嚢胞性線維症では、とにも免疫不全のがん、透析、および移植患者における多剤耐性を検討している。 P.における主要なMDRの排出ポンプ緑膿菌は、内膜プロトン-薬物アンチポーター（RND）、外膜のチャネル（OMF）、およびペリプラズムリンカー蛋白質（MFP）1-8から成る三者複合体である。 RNDとOMFタンパク質は、膜貫通蛋白質である。膜貫通型タンパク質は、全てのタンパク質の30％以上を構成し、現在の創薬ターゲットの65％です。疎水性の膜貫通ドメインは、水性緩衝液中のタンパク質が不溶性にします。膜貫通タンパク質を精製することができる前に、それは蛋白質が蛋白質の界面活性剤複合体（PDC）9月11日として可溶化できるように中性洗剤を含む緩衝条件を発見することが必要である。この例では、我々はRNDタンパク質、P.を使用してください、膜貫通タンパク質の組換え形態を表現洗剤を使用して、それを可溶化し、タンパク質の界面活性剤の複合体を精製する方法を示す緑膿菌の MexB貫通型トランスポーター、。この一般的な方法は、発現、精製、および他の多くの組換え的に発現する膜タンパク質の可溶化に適用することができます。タンパク質の洗剤の複合体は、後にX線結晶構造決定や架橋の研究を含む生化学的または生物物理学的特性評価に使用することができます。

Watch this Scientific Journal Video about Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein at JoVE.com

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

このプロトコルでは、可溶性タンパク質の洗浄剤複合体として、発現、可溶化、および組換え的に発現する膜蛋白質、MexBの精製を示す。 MexBは日和見細菌性病原体は緑膿菌からの多剤耐性膜輸送体である。

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

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

expression-detergent-solubilization-purification-membrane-transporter

Research

JoVE Journal

Biology

21.2K ビュー.  University of Illinois Chicago - UIC. このプロトコルでは、可溶性タンパク質の洗浄剤複合体として、発現、可溶化、および組換え的に発現する膜蛋白質、MexBの精製を示す。 MexBは日和見細菌性病原体は緑膿菌からの多剤耐性膜輸送体である。

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

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

結核および多剤耐性結核の診断のためのMODSの方法

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

生物学

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

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

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

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

蛍光プロテアーゼ保護を使用して膜タンパク質のトポロジの決定（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...