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>

<ol>
	<li>Wallin, E., von Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+integral+membrane+proteins+from+eubacterial,+archaean,+and+eukaryotic+organisms.">Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms.</a> Protein Sci. 7 (4), 1029-1038 (1998).</li><li>Fagerberg, L., Jonasson, K., von Heijne, G., Uhlen, M., Berglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+the+human+membrane+proteome.">Prediction of the human membrane proteome.</a> Proteomics. 10 (6), 1141-1149 (2010).</li><li>Okada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-Ray+diffraction+analysis+of+three-dimensional+crystals+of+bovine+rhodopsin+obtained+from+mixed+micelles.">X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles.</a> J Struct Biol. 130 (1), 73-80 (2000).</li><li>Palczewski, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+rhodopsin:+A+G+protein-coupled+receptor.">Crystal structure of rhodopsin: A G protein-coupled receptor.</a> Science. 289 (5480), 739-745 (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 (3), 289-298 (1996).</li><li>Wagner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+Escherichia+coli+for+membrane+protein+overexpression.">Tuning Escherichia coli for membrane protein overexpression.</a> Proc Natl Acad Sci U S A. 105 (38), 14371-14376 (2008).</li><li>Schlegel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+membrane+protein+overexpression+in+the+Escherichia+coli+strain+Lemo21(DE3).">Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3).</a> J Mol Biol. 423 (4), 648-659 (2012).</li><li>Chun, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+partner+toolchest+for+the+stabilization+and+crystallization+of+G+protein-coupled+receptors.">Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.</a> Structure. 20 (6), 967-976 (2012).</li><li>Drew, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-based+optimization+scheme+for+the+overexpression+and+purification+of+eukaryotic+membrane+proteins+in+Saccharomyces+cerevisiae.">GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.</a> Nat Protoc. 3 (5), 784-798 (2008).</li><li>Drew, D. E., von Heijne, G., Nordlund, P., de Gier, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+an+indicator+to+monitor+membrane+protein+overexpression+in+Escherichia+coli.">Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli.</a> FEBS Lett. 507 (2), 220-224 (2001).</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>Newstead, S., Kim, H., von Heijne, G., Iwata, S., Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescent-based+optimization+of+eukaryotic+membrane+protein+overexpression+and+purification+in+Saccharomyces+cerevisiae.">High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae.</a> Proc Natl Acad Sci U S A. 104 (35), 13936-13941 (2007).</li><li>Busso, D., et al. <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+protein+complexes+using+multiple+Escherichia+coli+protein+co-expression+systems:+a+benchmarking+study.">Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study.</a> J Struct Biol. 175 (2), 159-170 (2011).</li><li>Sonoda, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+membrane+protein+detergent+stability+for+improving+throughput+of+high-resolution+X-ray+structures.">Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.</a> Structure. 19 (1), 17-25 (2011).</li><li>Low, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analytical+gel+filtration+screening+of+integral+membrane+proteins+for+structural+studies.">High-throughput analytical gel filtration screening of integral membrane proteins for structural studies.</a> Biochim Biophys Acta. 1830 (6), 3497-3508 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>pOPIN-N-GFP</td>
			<td>addgene (www.addgene.org)</td>
			<td></td>
		</tr>
		<tr>
			<td>pOPINE-3C-GFP</td>
			<td>addgene (www.addgene.org)</td>
			<td></td>
		</tr>
		<tr>
			<td>C41(DE3)pLysS</td>
			<td>Lucigen (http://lucigen.com/</td>
			<td>60444-1</td>
		</tr>
		<tr>
			<td>LEMO21(DE3)</td>
			<td>New England Biolabs</td>
			<td>C2528H</td>
		</tr>
		<tr>
			<td>In-Fusion HD EcoDry Cloning System, 96 Rxns</td>
			<td>Clontech/Takara</td>
			<td>639688</td>
		</tr>
		<tr>
			<td>Power broth</td>
			<td>Molecular Dimensions (http://www.moleculardimensions.com/)</td>
			<td>MD12-106-1</td>
		</tr>
		<tr>
			<td>Protease Inhibitor tablets</td>
			<td>Roche</td>
			<td>11697498001</td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-Free; Protease Inhibitor Cocktail&#160;</td>
			<td>Roche</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>L-Rhamnose monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>83650</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P8849</td>
		</tr>
		<tr>
			<td>DNAse 1</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L7651</td>
		</tr>
		<tr>
			<td>Cholesterol hemisuccinate</td>
			<td>Sigma-Aldrich</td>
			<td>C6512</td>
		</tr>
		<tr>
			<td>CYMAL-6 ( 6-Cyclohexyl-1-Hexyl-&#946;-D-Maltoside)</td>
			<td>Anatrace</td>
			<td>C326</td>
		</tr>
		<tr>
			<td>DDM ( n-Dodecyl &#223;-maltoside )</td>
			<td>Generon</td>
			<td>D97002</td>
		</tr>
		<tr>
			<td>DM (n-Decyl &#223;-maltoside)</td>
			<td>Generon</td>
			<td>D99003</td>
		</tr>
		<tr>
			<td>LDAO ( N,N-Dimethyl-n-dodecylamine N-oxide)</td>
			<td>Generon</td>
			<td>D71111</td>
		</tr>
		<tr>
			<td>E1 ClipTip Eq384 8 channel 1-30 ul&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>4672030</td>
		</tr>
		<tr>
			<td>Rainin Pipette Pipet-Lite XLS 6ch 100-1200&#181;L LTS Spacer</td>
			<td>Anachem</td>
			<td>LA6-300XLS</td>
		</tr>
		<tr>
			<td>Rainin Pipette Light XLS+ 8ch 2-20&#181;L LTS</td>
			<td>Anachem</td>
			<td>L8-20XLSPLUS</td>
		</tr>
		<tr>
			<td>Pipette Pipet-Lite XLS 8ch 100-1200&#181;L LTS Tip</td>
			<td>Anachem</td>
			<td>L8-1200XLS</td>
		</tr>
		<tr>
			<td>24 well V bottom deep well blocks</td>
			<td>Porvair sciences</td>
			<td>360115</td>
		</tr>
		<tr>
			<td>BenchMark Fluorescent Protein Standard</td>
			<td>Life Technologies</td>
			<td>LC5928</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 10% Bis-Tris Midi Protein Gels, 26 well</td>
			<td>Life Technologies</td>
			<td>WG1203BOX</td>
		</tr>
		<tr>
			<td>XCell4 SureLock Midi-Cell</td>
			<td>Life Technologies</td>
			<td>WR0100</td>
		</tr>
		<tr>
			<td>Vibramax 100</td>
			<td>Heidolph</td>
			<td>544-21200-00</td>
		</tr>
		<tr>
			<td>Tension rollers attachment</td>
			<td>Heidolph</td>
			<td>549-81000-00</td>
		</tr>
		<tr>
			<td>ptima L-100 Ultracentrifuge running 45-Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>393253</td>
		</tr>
		<tr>
			<td>Optima Max-XP ultracentrifuge running TLA-55 rotor</td>
			<td>Beckman Coulter</td>
			<td>393315</td>
		</tr>
		<tr>
			<td>Floor standing centrifuge, Avanti J-26S XPI running JA-17 and JS-5.3 rotors&#160;</td>
			<td>Beckman Coulter</td>
			<td>B14535</td>
		</tr>
		<tr>
			<td>Prominence UFLC with RF-20A Fluorescence detector</td>
			<td>Shimadzu</td>
			<td></td>
		</tr>
		<tr>
			<td>Sepharose 6 10/300 GL size exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01&#160;</td>
		</tr>
		<tr>
			<td>SSI5R-HS SHEL LAB Floor Model Shaking Incubator, 5 Cu.Ft. (144 L), 30-850 RPMs</td>
			<td>Shel Lab&#160;</td>
			<td>SSI5R-HS</td>
		</tr>
		<tr>
			<td>Innova44R incubator</td>
			<td>New Brunswick /Eppendorf</td>
			<td>Innova44R</td>
		</tr>
		<tr>
			<td>TS SERIES 0.75KW DISRUPTER 230V 50HZ 1PH (40KPSI)</td>
			<td>Constant systems</td>
			<td>PL083016</td>
		</tr>
		<tr>
			<td>L-Rhamnose monohydrate</td>
			<td>Sigma</td>
			<td>83650</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 ...

green-fluorescent-protein-based-expression-screening-membrane

Research

JoVE Journal

Biology

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

32.4K 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

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.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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

科研

生物学

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

在油脂介孔膜蛋白的结晶

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...