Name: expression, detergent solubilization, and purification of a membrane transporter, the mexb multidrug resistance protein
Uploaded: 2010-12-03T12:00:00
Description: Watch this Scientific Journal Video about Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein at JoVE.com

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?...

<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. Agents Chemother. 38, 1742-1752 (1994).</li><li>Li, X. Z., Nikaido, H., 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=Role+of+MexA-MexB-OprM+in+antibiotic+efflux+in+Pseudomonas+aeruginosa.">Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa.</a> Antimicrob. Agents Chemother. 39, 1948-1953 (1995).</li><li>Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H., Nishino, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+specificities+of+MexAB-OprM,+MexCD-OprJ,+and+MexXY-oprM+efflux+pumps+in+Pseudomonas+aeruginosa.">Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa.</a> Antimicrob. 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. Crystallogr. 61, 724-730 (2005).</li><li>Jones, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactants+in+membrane+solubilisation.">Surfactants in membrane solubilisation.</a> Int. J. Pharm. 177, 137-159 (1999).</li><li>Maire, M. l. e., Champeil, P., Moller, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+membrane+proteins+and+lipids+with+solubilizing+detergents.">Interaction of membrane proteins and lipids with solubilizing detergents.</a> Biochim. Biophys. Acta. 1508, 86-111 (2000).</li><li>Miroux, B., Walker, J. 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>

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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		<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 ...

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

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 ...

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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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÃƒÅ½Ã¢â‚¬Â41

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

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

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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

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

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  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

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

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

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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 ...