Name: green fluorescent protein-based expression screening of membrane proteins in escherichia coli
Uploaded: 2015-01-06T11:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli at JoVE.com

The OPPF-UK is funded by the Medical Research Council, UK (grant MR/K018779/1) and the Membrane Protein Laboratory by the Wellcome Trust (grant 099165/Z/12/Z).

1.构建表达载体<ol><li>使用的PCR输液克隆构建高达96表达质粒并联使用矢量pOPINE-3C-GFP和POPIN-N-GFP其中或者可分解的C末端或N末端 ​​的GFP融合的His6添加到分别序列13。 注意:载体的选择是由该蛋白质的拓扑口授由于GFP的需要被表达在胞质溶胶中，从而用于与胞质N末端 ​​和细胞外C末端，我们将使用POPIN-N-GFP和用于蛋白质细胞外N端和胞质C端的，我们会用pOPINE-3C-GFP。其中，两个末端是胞质我们将使用pOPINE-3C-GFP除非有官能理由不标记的C-末端。 </li><li>扩增用掺入所示的下列引物扩展编码膜蛋白的DNA序列: pOPINE-3C-GFP（正向引物 - AGGAGATATACCATG;反向底漆 - CAGAACTTCCAGTTT）和人口信息网-N-GFP（正向引物 - AAGTTCTGTTTCAGGGCCCG;反向底漆 - ATGGTCTAGAAAGCTTTA）。 </li><li>分析用琼脂糖凝胶电泳PCR反应。一旦清洁的PCR产物已经获得，添加5单位DPNI至每孔（补于5μl1X DPNI反应缓冲液）。纯化使用磁珠在96孔格式的PCR产物。 注意:DPNI加入以除去模板矢量;此步骤不是必要的，如果基因组DNA用作模板。 </li><li>加入1μl适当线性化载体（100毫微克）与PCR板的每个孔中。 </li><li>使用多通道移液器加X微升纯化PCR的插入到PCR板的合适的孔中。确保所有的产品都在10-250纳克的范围内。 </li><li>加（9-x）的微升水到井和整个10微升转移到含有干式中融合试剂的管。离开30秒。 </li><li>吹打向上和向下短暂混合内容。转让反应回原始PCR板和密封。 </li><li>在热循环仪孵育30分钟，在42℃。 </li><li>立即一旦反应转移到冰作为反应完全，并添加40微升的TE（10毫摩尔Tris，pH值8.0，1mM EDTA）中。 </li><li>冻结在一次或转化为能力的细胞。对于转型，用3微升稀释反应，每高能力的50微升等分（&gt; 5×10 9转化子/微克）主管E.大肠杆菌细胞。 </li><li>加入1毫升的LB琼脂补充有50微克/毫升羧苄青霉素每孔的24孔组织培养板（用于克隆，制备孔为2×构建体的数量）。 </li><li>下列产物，继生长板出25微升和25微升1 5稀释的培养物的上含24孔组织培养板中的琼脂上。 </li><li>轻轻倾斜盘传播的细胞。 </li><li>干燥该板与盖关闭10-15分钟，在37℃或在FLO瓦特罩在室温下。 </li><li>更换盖子，把板倒过来，孵育过夜，37℃。 </li><li>选择两个殖民地和微型准备的载体。 </li><li> PCR验证使用构建体特异性反向引物和载体特异性正向引物（ 例如 pOPIN_FOR GAC CGA AAT TAA TAC GAC TCA CTA TAG GG）的构建体。 </li></ol> 2.转换E的Ë大肠杆菌菌株XPRESSION 注:在执行该协议，E.大肠杆菌感受态细胞制造的，等分并存储为先前13描述的冷冻在-80℃。膜蛋白表达常规筛选在两株，Lemo21（DE3）和C41（DE3）pLysS中。为了有助于对结果的解释也可以是有帮助的，包括一个阳性对照， 例如 ，一个质粒表达GFP或融合于GFP的膜蛋白已知表达和空载体的阴性对照。 <ol><li>解冻的阿里quotted E.大肠杆菌在冰上。加入3微升迷你坦然表达质粒的感受态细胞的各等分试样。 </li><li>冰上孵育混合30分钟。 </li><li>在42℃的热休克的细胞，持续30秒。 </li><li>使细胞恢复在冰上2分钟，然后添加300微升的功率肉汤到每个管中。 </li><li>孵育1小时在37℃静态孵化器。 </li><li>制备的LB琼脂在24孔组织培养板，补充有50微克/毫升羧苄青霉素和35微克/毫升氯霉素。添加鼠李至0.25mm的最终浓度，以含有Lemo21（DE3）中的孔中。 注意:如果该产品可能是有毒的含C41的孔（DE3）pLysS中可含1％（重量/体积）葡萄糖的补充。 </li><li>转移30微升来自转化混合物中的细胞上的LB琼脂板上。传播和在1.13-1.15描述干燥板。 </li></ol> 3.制备隔夜发酵剂注:始终改造后从来没有从旧改造板准备过夜培养的一天。 <ol><li>添加0.7毫升功率肉汤到96孔深孔板补充有50微克/毫升羧苄青霉素和35微克/毫升氯霉素的每个孔中。添加鼠李含有Lemo21（DE3）中的孔中，以0.25mm的最终浓度。任选地，补充C41（DE3）pLysS中，用1％（重量/体积）葡萄糖;见上文（2.6注）。 </li><li>从隔夜转化平板在每孔中选择一个殖民地。密封与透气的密封块。 </li><li>摇该块以250rpm在具有大掷（ 例如 25毫米的轨道），或以600rpm在培养箱具有小掷（ 例如，一个4.3毫米轨道），在37℃过夜的培养箱。 </li></ol> 4.生长和诱导文化<ol><li>加3毫升功率肉汤补充有50微克/毫升羧苄青霉素和35微克/毫升氯霉素的每个瓦特ELL的24孔深孔板。添加L-鼠李糖（葡萄糖），如第3.1节所述。用3毫升培养，以允许收集一式两份，并允许水穿过气体可渗透密封部分蒸发。 </li><li>通过加入150微升Lemo21（DE3）或C41（DE3）pLysS中过夜培养物的24孔深孔块的每个孔设置的表达培养物。 </li><li>摇动，在37℃的块，直到在595nm处的平均外径为0.5〜穿过板。 </li><li>通过加入IPTG到培养至1.0毫IPTG的终浓度诱导表达。 </li><li>生长培养过夜（18-20小时）振荡在20℃。 </li></ol>表达了在凝胶荧光5.分析<ol><li>收获一式两份。传送1毫升培养的从每个孔成正方形井，锥形底，96孔深孔块。 注意:收获在重复的情况下的块或破损的，以允许alternati的测试如果需要的话已经洗涤剂。 </li><li>密封块并通过离心收获细胞，在6000×g离心10分钟。 </li><li>反转块，倒出媒体。轻轻敲击块到纸巾排出剩余的介质。 </li><li>密封该板，并储存在-80℃下进行至少20分钟。任选地，离开该实验在这一点上与在处理时方便该块。 </li><li>去霜粒料进行20分钟，在室温下进行。 </li><li>使用一个多通道移液器或轨道摇床（1000转，10分钟），在4℃以完全悬浮细胞在200μl的裂解缓冲液（50mM的NaH 2 PO 4，300mM NaCl的10mM咪唑1％体积/ V吐温20，pH调节至8.0用NaOH）。 </li><li>加入20微升的DLPI溶液（20微升DNA酶I（4000单位/ ml），20毫克溶菌酶，和20微升蛋白酶抑制剂混合物在2毫升水中的终体积）。 10分钟孵育在4℃下振荡（〜1000转）。 </li><li&gt;添加25微升10％ 正十二烷基β-D-麦芽糖苷（DDM）。 </li><li> 60分钟孵育在4℃下振荡（〜1000转）。 </li><li>离心机的深阱块在6000×g离心30分钟，在4℃。 </li><li>转移10微升澄清的裂解液的一种微量滴定板，并添加10微升的SDS PAGE凝胶上样缓冲液（100毫摩尔Tris，pH值6.8，4％w / v的SDS，0.2％w / v的溴酚蓝，10％体积/体积β巯基乙醇，和20％体积/体积的甘油）。 小心！不要煮沸样品中 。 </li><li>负载10微升样品从步骤5.11 /孔到SDS-PAGE凝胶（S）和在100-120 V（恒定电压）在冷室中运行，直到染料前沿到达底部（2-2.5小时）。 </li><li>将凝胶上的成像器（ 例如，用蓝色光滤波器来检测GFP融合蛋白）。被选择的曝光时间，以确保最亮的带不饱和的。 </li></ol> 6.规模化细胞培养<ol><li>变换主管等分<em&gt;电子。大肠杆菌在第2条所描述的。 </li><li>选择一个殖民地到10ml的电源肉汤（如第3条所述补充），并在37℃生长过夜。 </li><li>稀释将5ml过夜培养物到500ml功率肉汤。 </li><li>成长带以250rpm摇动，在37℃，直到OD在595nm处为〜0.5。 </li><li>通过加入IPTG至1.0mM的终浓度诱导表达。 </li><li>与以250rpm振荡在20℃下生长过夜。 </li><li>收获细胞，离心，在5000×g离心15分钟。 </li><li>弃去上清液。细胞沉淀可存放在-80℃。 </li></ol> 7.准备膜的<ol><li>解冻细胞（如有必要）在每克细胞沉淀5毫升的体积室温并重新悬浮于1×PBS中的pH 7.5;增加音量必要的，直到粘度降低到流的一致性。添加的MgCl 2至1mM，DNA酶I的最终浓度为10微克/毫升和1终浓度预包装的每20克细胞沉淀的蛋白酶抑制剂片剂。打破使用冷冻细胞破碎开放单元在30 kpsi的压力下进行。 </li><li>沉淀不间断细胞和碎片以30,000离心15分钟，在4℃。转移上清液至一个超离心管中。 </li><li>收集通过降低盘片在超离心机将上清液以200,000×g离心1小时，在4℃的总的膜级分。 </li><li>弃去上清液。 </li><li>使用细胞匀浆重新悬浮膜沉淀在冰冷的10毫升的PBS pH为7.5。 1毫升将再悬浮的膜所需要的各洗涤剂。任选地，在此阶段，闪存冻结的膜悬浮液在液氮中，并储存在-80℃。 </li></ol> 8.溶解膜组分的分析<ol><li>用于增溶融膜部分（如果有必要），并且对于每个洗涤剂，等份加入900μl膜悬浮液的进1.5ml的异质同晶微centrifuGE管。 </li><li>加入100微升每新鲜制备的洗涤剂（10％重量/ DDM，DM诉溶液，Cymal-6和LDAO），以1％的终浓度，以指定的1.5mL管中。对于真核细胞膜蛋白胆固醇半琥珀酸酯（0.2％最终浓度）溶解，可以加入到洗涤剂中。 </li><li>孵育混合物在4℃下1小时以温和搅动。 </li><li>沉淀洗涤剂不溶部分用台式超离心机，确保管是至少半满，在150000克在4℃下45分钟，并保留上清液。 注意:洗涤剂增溶的有效性可以使用荧光读板器通过测量溶解的膜与悬浮于PBS中的相同体积的洗涤剂不溶性级 ​​分的GFP荧光来估计。 </li><li>分析单分散度不同的洗涤剂:使用荧光检测的尺寸排阻色谱法的膜蛋白复合物（FSEC）。 </li><li>对于FSEC分析，达到平衡尺寸排阻柱具有宽分馏范围， 例如 5000至5,000,000的分子量，以在0.3毫升/ min流速运行缓冲液（20mM的Tris pH值7.5，150 mM氯化钠，0.03％，DDM） 。使用这个缓冲区所有样品，也就是说，它不改变对每个洗涤剂测试。 </li><li>注入100微升溶解的膜在柱上以0.3毫升/分钟的流速。监视使用荧光光学元件（激发在488nm和在512纳米发射）洗脱曲线。如果内嵌荧光显示器不可用，收集级分并在读板读数器的荧光。 </li><li>进口洗脱体积和荧光强度数据转化为图形显示的电子表格程序。 </li><li>分析在第5节由凝胶荧光剩余溶解膜材料。 </li></ol>

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The authors have nothing to disclose.

在这篇文章中描述的协议，结扎独立克隆到GFP报告载体是结合荧光检测来快速筛选在大肠杆菌中的多个膜蛋白的相对表达大肠杆菌 。向量可以在四个工作日内与主要表达筛选采取进一步的一周进行。在凝胶全细胞裂解物的洗涤剂的荧光被用来排名命中表达进行进一步的分析中的二次屏幕。作为呈现的主要表达屏幕不需要任何专门设备除了适用于深井块生长细胞摇床培养箱。所有其他液体处理涉及的96孔平板的SBS可以使用多通道移液器进行。该协议可以很容易地修改，以包括不同的表达的条件（ 例如较低的温度）下生长的其他大肠杆菌菌株。在LEMO21（DE3）中的情况下，滴定鼠李的水平（从0至2.0毫摩尔）加到调节转录的速率可以增加一些膜蛋白7的表达水平。洗涤剂比DDM其它可用于提取在主屏幕虽然苛刻洗涤剂会从包涵体溶解蛋白质，因此给予误报。显然，更详细说明的初始画面变得越费时的实验。 次级屏幕涉及制备从在初级筛选中鉴定的表达命中一个总的膜级分。这是一个关键步骤，因为这将证实该融合蛋白的定位，以大肠杆菌的膜大肠杆菌细胞。的GFP融合蛋白在不同洗涤剂随后提取通过荧光检测到的大小排阻溶化材料的色谱监测，以评估单分散度洗涤剂 - 蛋白质复合物的。这又是一个关键的一步，因为它确定最合适的洗涤剂增溶的膜蛋白进行随后的下游加工。然而，经验表明，可能仍纯化和结晶期间被要求的洗涤剂（多个）的选择的进一步优化。在不同的去污剂包括那些具有相对较小胶束粒度（ 例如 LDAO）均匀行为证据似乎是一种倾向，以形成良好的晶体衍射至少为原核生物膜蛋白14的良好指标。在这方面示出用于图2B中的膜蛋白的轮廓似乎非常有利。 使用GFP报告的主要优点是，它提供了快速读出表达的未纯化的样品中。一个缺点是，在GFP融合蛋白具有的蛋白质进行结晶的纯化过程中被除去。在这里使用的载体的情况下，一个鼻病毒3C蛋白酶位点使GFP的裂解。可替代地，它是直接重新克隆候选蛋白，在筛选中鉴定，到载体中仅添加一个聚组氨酸或其它亲和标记为随后的纯化。这假定融合到绿色荧光蛋白是没有必要的表达。主替代使用融合到绿色荧光蛋白检测膜蛋白表达和增溶的是只添加组氨酸标签。然而，这种方法通常需要开始一个膜部分15，它为许多变体的评价将变得非常费时。 总之，本文中描述的协议的主要目的是使大量的膜蛋白序列的变体可以迅速地评价在表达和洗涤剂增溶的术语和优先进行更深入的分析。

据估计，膜蛋白占编码所有测序的基因组1，包括人类基因组2的基因的约20-30％。定在许多生物过程的关键作用，对于代谢产物，能产生例如运输和作为药物靶标，存在在确定其三维结构的强烈兴趣。然而相比可溶性蛋白，膜蛋白是高度低于限额在蛋白质数据库。在99624释放结构在PDB（在实际上， <a href="http://www.rcsb.org/pdb/">http://www.rcsb.org/pdb/</a> ）只有471（ <a href="http://blanco.biomol.uci.edu/mpstruc/">http://blanco.biomol.uci.edu/mpstruc/</a> ）被分类为膜蛋白。这反映了与它们天然表达在相对低的水平的膜蛋白加工的技术困难;视紫红质是少数例外3,4之一。过度表达的重组版本S IN的异源细胞经常导致错误折叠和差的定位，以限制了生产的总体水平膜上。选择用于提取膜蛋白和溶解的最佳洗涤剂一旦表达使得随后的纯化困难并且需要仔细的优化。 大肠杆菌仍然是膜蛋白占72％（最常用的表达宿主<a href="http://blanco.biomol.uci.edu/mpstruc/">http://blanco.biomol.uci.edu/mpstruc/</a>结构）解决迄今这是几乎全部来自细菌来源。具体E.大肠杆菌菌株，C41（DE3）和C43（DE3）中，已分离出不导致过度表达膜蛋白，从而避免毒性观察其他BL21菌株5,6的影响。调谐重组膜蛋白表达的水平似乎是优化生产6,7的水平是至关重要的。 6-8的评价。因此，对于多个膜蛋白平行的表达筛选的方法是至关重要的。一种常用的方法是将融合蛋白为绿色荧光蛋白（GFP）使表达，而不需要纯化蛋白进行跟踪。以这种方式，关于表达量，稳定性和行为在不同洗涤剂信息可与少量的未纯化的材料9-12中进行评估。 在这篇文章中，我们描述了一个精简的协议膜蛋白在大肠杆菌中克隆和表达筛选利用大肠杆菌融合到GFP的生产和洗涤剂随后表征记者:蛋白质补偿LEXES（ 图1）。

<table><tbody><tr><td>pOPIN-N-GFP</td><td>addgene (www.addgene.org)</td><td></td><td></td></tr><tr><td>pOPINE-3C-GFP</td><td>addgene (www.addgene.org)</td><td></td><td></td></tr><tr><td>C41(DE3)pLysS</td><td>Lucigen (http://lucigen.com/)</td><td>60444-1</td><td></td></tr><tr><td>LEMO21(DE3)</td><td>New England Biolabs</td><td>C2528H</td><td></td></tr><tr><td>In-Fusion HD EcoDry Cloning System, 96 Rxns</td><td>Clontech/Takara</td><td>639688</td><td></td></tr><tr><td>Power broth</td><td>Molecular Dimensions (http://www.moleculardimensions.com/)</td><td>MD12-106-1</td><td></td></tr><tr><td>Protease Inhibitor tablets</td><td>Roche</td><td>11697498001</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-Free; Protease Inhibitor Cocktail&amp;nbsp;</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>L-Rhamnose monohydrate</td><td>Sigma-Aldrich</td><td>83650</td><td></td></tr><tr><td>Protease Inhibitor Cocktail&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8849</td><td></td></tr><tr><td>DNAse 1</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L7651</td><td></td></tr><tr><td>Cholesterol hemisuccinate</td><td>Sigma-Aldrich</td><td>C6512</td><td></td></tr><tr><td>CYMAL-6 (6-Cyclohexyl-1-Hexyl-&amp;beta;-D-Maltoside)</td><td>Anatrace</td><td>C326</td><td></td></tr><tr><td>DDM (n-Dodecyl &amp;beta;-maltoside)</td><td>Generon</td><td>D97002</td><td></td></tr><tr><td>DM (n-Decyl &amp;beta;-maltoside)</td><td>Generon</td><td>D99003</td><td></td></tr><tr><td>LDAO (&lt;em&gt;N,N&lt;/em&gt;-Dimethyl-n-dodecylamine &lt;em&gt;N&lt;/em&gt;-oxide)</td><td>Generon</td><td>D71111</td><td></td></tr><tr><td>E1 ClipTip Eq384 8&amp;nbsp;channel 1-30 &amp;micro;l</td><td>Thermo Scientific&amp;nbsp;</td><td>4672030</td><td></td></tr><tr><td>Rainin Pipette Pipet-Lite XLS 6ch 100-1,200 &amp;micro;l LTS Spacer</td><td>Anachem</td><td>LA6-300XLS</td><td></td></tr><tr><td>Rainin Pipette Light XLS+ 8ch 2-20 &amp;micro;l LTS</td><td>Anachem</td><td>L8-20XLSPLUS</td><td></td></tr><tr><td>Pipette Pipet-Lite XLS 8ch 100-1,200 &amp;micro;l LTS Tip</td><td>Anachem</td><td>L8-1200XLS</td><td></td></tr><tr><td>24 well V bottom deep well blocks</td><td>Porvair sciences</td><td>360115</td><td></td></tr><tr><td>BenchMark Fluorescent Protein Standard</td><td>Life Technologies</td><td>LC5928</td><td></td></tr><tr><td>NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 26 well</td><td>Life Technologies</td><td>WG1203BOX</td><td></td></tr><tr><td>XCell4 SureLock Midi-Cell</td><td>Life Technologies</td><td>WR0100</td><td></td></tr><tr><td>Vibramax 100</td><td>Heidolph</td><td>544-21200-00</td><td></td></tr><tr><td>Tension rollers attachment</td><td>Heidolph</td><td>549-81000-00</td><td></td></tr><tr><td>ptima L-100 Ultracentrifuge running 45-Ti rotor</td><td>Beckman Coulter</td><td>393253</td><td></td></tr><tr><td>Optima Max-XP ultracentrifuge running TLA-55 rotor</td><td>Beckman Coulter</td><td>393315</td><td></td></tr><tr><td>Floor standing centrifuge, Avanti J-26S XPI running JA-17 and JS-5.3 rotors&amp;nbsp;</td><td>Beckman Coulter</td><td>B14535</td><td></td></tr><tr><td>Prominence UFLC with RF-20A Fluorescence detector</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Sepharose 6 10/300 GL size exclusion column</td><td>GE Healthcare</td><td>17-5172-01&amp;nbsp;</td><td></td></tr><tr><td>SSI5R-HS SHEL LAB Floor Model Shaking Incubator, 5 Cu.Ft. (144&amp;nbsp;L), 30-850 rpm</td><td>Shel Lab&amp;nbsp;</td><td>SSI5R-HS</td><td></td></tr><tr><td>Innova44R incubator</td><td>New Brunswick /Eppendorf</td><td>Innova44R</td><td></td></tr><tr><td>TS Series 0.75 kW Disrupter 230 V 50 Hz 1&amp;nbsp;PH (40 kpsi)</td><td>Constant systems</td><td>PL083016</td><td></td></tr><tr><td>L-Rhamnose monohydrate</td><td>Sigma</td><td>83650</td><td></td></tr></tbody></table>

green fluorescent protein-based expression screening of membrane proteins in escherichia coli

重组膜蛋白结构和功能研究的生产仍然在技术上具有挑战性的由于表达水平低，许多膜蛋白的固有的不稳定性，一旦溶解在洗涤剂。的协议描述，结合膜蛋白结扎独立克隆的GFP融合在了GFP荧光检测大肠杆菌表达。这使得多个膜蛋白的结构和表达筛选/变种，以找出适合的时间和精力进一步投资的候选人。 GFP报告是由可视化的GFP荧光下列SDS聚丙烯酰胺凝胶电泳（SDS-PAGE），用在表达的一个主屏幕。膜蛋白，显示都与最小退化高表达水平由于缺乏自由绿色荧光蛋白的所指示的，被选择为辅助屏幕。这些构建体被定标和一个总的膜级分制备和增溶D在四种不同的洗涤剂。以下超速离心以除去去污剂不溶性材料，裂解物通过荧光检测尺寸排阻色谱法（FSEC）分析。监控尺寸排阻轮廓通过GFP荧光提供了关于在不同洗涤剂的膜蛋白的单分散度和完整性信息。蛋白质:与一个对称峰具有很少或没有自由GFP和最小聚合洗脱洗涤剂组合候选为随后的纯化。使用上述方法，所述的异源表达在大肠杆菌中的SED（形状，伸长，除法和孢子）从47个不同种类的细菌蛋白质的大肠杆菌进行了分析。这些蛋白质通常具有10个跨膜结构域，并且是​​细胞分裂至关重要。结果表明，生产的在大肠杆菌中的直向同源物的SED 大肠杆菌是用GFP融合蛋白的相对表达水平和完整性高度可变。实验我dentified进一步调查的一个子集。

所述SEDS家族蛋白（形状，伸长率，除法和孢子）是用于细菌细胞分裂至关重要。这些积分蛋白通常具有与细胞内两者的氨基和羧基末端10个跨膜结构域。为了举例说明上述的协议，47的SED直向同源物被克隆到载体pOPINE-3C-EGFP。所述构建体的小规模表达屏幕进行在二号E .coli菌株Lemo21（DE3）中的0.25毫摩尔的存在下生长鼠李阻止泄露表达和C41（DE3）pLysS中，这是常规用于膜中的表达蛋白质5-8。 诱导培养物的洗涤剂溶解裂解物通过SDS-聚丙烯酰胺电泳分析。使用凝胶荧光可视化表达GFP融合蛋白表明，两株之间38出47的SED结构的制作。有趣的是有两个表达菌株之间的差异。的38表示的结构中，只有18人在生产两种菌株，14只仅在C41（DE3）pLysS中表达Lemo21（DE3）和6。总体而言，有较高的成功率的Lemo21（DE3）相比，C41（DE3）pLysS中（38比24）。然而，一些蛋白质似乎是应变具体观察意味着筛选在两个是有用的。 为47的SED中的24表示数据结构示于图1中 。所述条带的强度给出的指示表达水平的， 例如 ，在井C4在图1A和1B的融合蛋白被表达在两个菌株然而附加较低分子量带表明所述蛋白质是部分降解。在许多泳道有对应于大小的GFP单独的频带。的蛋白质的完整的定性评估可以如下进行:在寻找相对于自由GFP融合蛋白的强度;如:G4和H4被完全分解为在C41（DE3）pLysS中（ 图1B）的分类的GFP，而在Lemo21（DE3）中有一些完整的蛋白质（ 图1A）。为14列的38个表达的蛋白质的游离的GFP带的强度相似，融合蛋白，对于21自由绿色荧光蛋白带的强度大于所述融合蛋白和融合蛋白的一小部分（3 / 38表示）， 例如 F6和G6在图1A中有相对小的自由绿色荧光蛋白相比，融合蛋白。 二，在主屏幕B5表达良好（高强度带自由绿色荧光蛋白和融合蛋白）和G6（小分类GFP）的表达，并从500 ml细胞培养物制备总膜部分的构造。将再悬浮的膜被分成等份并通过荧光检测到的尺寸排阻色谱法（FSEC）分析。所述FSEC剖面，在造型玩具呈现分别以及e 2A和2B表明，这两种蛋白质的SED的行为不同的四个洗涤剂测试。在一个案例中（ 图2A:样品B5）的蛋白质只给了一个对称峰与DDM小聚集，因此这将是首选的清洁剂进行后续的纯化。相比之下，其他的融合蛋白（ 图2B:样品G6），得到在所有测试的洗涤剂的类似信息。 <img alt="图1" src="/files/ftp_upload/52357/52357fig1highres.jpg" width="700px" /> 图1:图的工作流程的用于在大肠杆菌中筛选膜蛋白表达使用大肠杆菌 GFP的记者。 <img alt="图2" src="/files/ftp_upload/52357/52357fig2highres.jpg" /> 图2:膜蛋白的表达使用凝胶荧光主屏幕。 </strong&gt;洗涤剂E.裂解液大肠杆菌细胞通过SDS-PAGE进行了分析和凝胶使用蓝色的Epi照明和一个二十八分之五百三十〇滤波器成像。其结果示于24构建体（基于其在96孔板位置标记的A4-H6）。的频带进行自由GFP和GFP融合蛋白的位置来表示。（A）中表示的Lemo21（DE3）（B）中表示在C41（DE3）pLysS中蛋白质蛋白质。 <img alt="图3" src="/files/ftp_upload/52357/52357fig3highres.jpg" /> 图3:膜蛋白表达的使用荧光检测的尺寸排阻色谱（FSEC）辅助屏幕的总的膜级分分别提取在四个不同的清洁剂和溶解的膜蛋白通过尺寸排阻色谱使用耦合到荧光检测器的FPLC系统进行分析。对于FSEC型材（A）的膜蛋白B5和（B）的 </s仲&gt;膜蛋白G6。对应于聚集蛋白，溶解GFP融合蛋白和游离的GFP的峰的位置表示。测试的洗涤剂如下所示的配置文件。

Watch this Scientific Journal Video about Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli at JoVE.com

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

简化的方法来筛选重组膜蛋白的基础上融合绿色荧光蛋白大肠杆菌表达呈现。

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

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

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

32.7K 次观看.  Research Complex at Harwell. 简化的方法来筛选重组膜蛋白的基础上融合绿色荧光蛋白大肠杆菌表达呈现。 

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

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

科研

生物化学

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.

A construct encoding TMEM184A with a GFP tag at the carboxy-terminus designed for eukaryotic expression, was employed in assays designed to confirm the identification of TMEM184A as a heparin receptor in vascular cells.

用GFP标记TMEM184A构建肝素受体身份确认

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

遗传学

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

化学

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

通过荧光体积排阻色谱快速评估膜蛋白质量

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

用质谱和荧光法研究非规范氨基酸和蛋白质分析的选择性压力下工程 "金" 荧光

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

生物工程

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

免疫与感染

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition 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.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

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

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

膜蛋白的绿色荧光蛋白为基础的表达筛选

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