作者感谢美国国立卫生研究院（AI074224和GM085323）和美国国家科学基金会（0712019和0924699）提供资金支持项目，致力于异源生物合成。

下面的文字是红霉素A抗生素的具体的功能，但该步骤被设计成一般适用于异源生物合成的其它天然产物的作为候选。 1。红霉素的遗传集群式转移<ol><li>设计PCR引物扩增的所有的相关的基因的红霉素A群集内的S.红霉素染色体。这一步是针对那些自然的候选产品，其基因序列已被确定。或者，所需的基因，可以合成（通过几个商业供应商选项）的优点，新合成的基因将被设计为最佳的密码子的使用范围内的新的大肠杆菌大肠杆菌宿主。如果DNA合成的追求，目前的挑战是的产生megasynthase基因的长度可能超过10 KB。技术上，合成可以完成，但它是具有挑战性的和昂贵的。可以预料，不断improvem已废除的基因合成技术，将解决目前的缺点，进一步验证这种方法提供外源遗传物质。 </li><li>使用新鲜配制S.进行PCR 红霉素的基因组DNA作为模板。的染色体DNA，可以从培养的S.制备红霉素或从冻存的细菌（ 图3）25。 PCR反应可能受益于加入DMSO（4微升在50μl的反应）考虑到在目标中的G+ C含量高S。红霉素基因。应每个扩增的​​基因测序，以确认，完整的基因已被检索和没有突变的PCR过程中已被引入。 </li><li>通过限制性酶切和结扎，每一个孤立的基因插入到表达载体设计的E.大肠杆菌 （ 图3）。这是由在PCR引物设计酶切位点，让消化和连接到合适的防爆的气缸压力载体（包含在图3的例子进行了）。我们已经用pET21，pET28载体。 pET28载体允许包含计算机辅助基因表达的红霉素A的5&#39;前导序列。确认个别基因的表达，通过逆转录聚合酶链反应（RT-PCR），SDS-PAGE分析（使用可溶性蛋白质级分），或方便的表型分析（ 图4）。 </li><li>构建根据从个别的表达质粒（ 图3）中成功表达基因的操纵子。传统上，我们已使用兼容粘性的限制性内切酶（如的XbaⅠ和 SpeⅠ）24的组合，顺序地转换基因的操纵子，然而，为操纵子施工选择的限制性位点，必须不驻留在（或必须删除从）的孤立基因序列（如果使用基因合成可避免的问题）。这一步巩固了20红霉素A基因之前，E.大肠杆菌变换，细则第十五。目前，我们已经用这种方法包括了20 kb的外源DNA导入一个标准的pET载体（2〜10 kb的基因）。此外，我们还用这种方法来生成包含9个基因的操纵子。它是未知的到底有多少DNA或可以被集成到多少基因的pET-型质粒，但是，以上专利的度量为红霉素生物合成途径的复杂的系统，如提供一个参考。最后，标准在体外连接步骤，并随后成功转型的步骤变得越来越困难，因为操纵子的质粒大小的增加。结扎的温度（12-22℃）和时间（3-24小时）是多种多样的和电变换被用来帮助最终的质粒构建体的制造过程。 </li><li>全新设计的生物合成质粒转化到E.大肠杆菌菌株BAP1使用电转化（ 图5）。该过程可以尝试用质粒引入按顺序或合作mbination。当尺寸能够或大量的质粒转化，我们传统上使用的电转化（如相对的化学转化）。一旦转化，板的固体LB培养基中的细胞在含四环素（5毫克/升），羧苄青霉素（100毫克/升），卡那霉素（50毫克/升），链霉素（50毫克/升），和安普霉素（50毫克/升）选择质粒的维护。这是极不寻常的使用六种不同的选择抗生素后转型的E.大肠杆菌细胞，但是这只是反映允许最终红霉素A的生物合成所需要的质粒的总数。虽然有可能，该方法将限制一次培养已开始（请参见下文）的化合物的生产能力。 </li></ol> 2，E.大肠杆菌生物合成重组<ol><li>应变BAP1已设计，以提供所需的底物的生物合成，并以发布翻译修改deoxyerythronolide乙megasynthase，。个人的eryt基因hromycin的通路已测试的基因表达和巩固操纵子到原核表达质粒。本节专门培养条件测试的全红霉素A通路的共同行为。 </li><li>的新鲜转化BAP1以3个独立的菌落，接种1.5毫升LB培养含有所需质粒选择抗生素（上面所指出，在固体培养基上选择转化体）在相同浓度的异丙基β-D-1-硫代吡喃半乳糖苷（IPTG; 100μM ）和阿拉伯糖（2毫克/毫升），以诱导基因的表达，和20mM丙酸钠以支持细胞内的生物合成的前体形成。一旦生产已得到确认，可进行更大的文化，企图扩展和最大限度地提高产品效价。 </li><li>培养细胞在22°C和250转每分钟24〜48小时。在这种情况下，抗生素的选择确保了所需的质粒被保持在细胞培养的早期阶段。然而，质粒作为选择抗生素的水平发生变化的过程中在培养期间，s可能很容易丢失。这可能会导致质粒不稳定的（ 即在培养过程中的一个或多个质粒的损失），这将限制产能。当不相容质粒被用来引入一个完整的异源基因簇，该问题更加恶化。就是这样的情况下，的红霉素A化合物（ 图5），就必须真正地纠正过量的化合物E.大肠杆菌 。然而，证明初始生产的目的，这样的设计上的缺陷被容忍为了尽快建立的异源的方法的可行性。 </li><li>在10,000 rpm下在Eppendorf离心澄清，通过离心分离的文化。除去上清液并储存在4℃下进行分析。 </li><li>红霉素A的情况下，缺乏化合物生产处理列入GroES / EL伴侣蛋白（使用pGro7专门为解决缺乏活动的酶表达后），并列入额外的基因拷贝（这些基因涉嫌弱表达/活动）。类似的策略可能需要与其他异源天然产品的生产工作。 </li></ol> 3。产品分析<ol><li>确认并量化红霉素A的形成，注入的培养上清到一个LC-MS系统（ 图6）。市售红霉素A被用来确认生产和作为量化过程中的一个标准。对于没有商业上可用的标准的情况下，将需要额外的LC-MS分析化学NMR和高分辨质谱表征。 </li><li>与生产之间的3个菌落/文化BAP1转型。选择最高的生产商和一个甘油从殖民地源的准备。此步骤可以并行完成议定书段的生产培养2通过使用培养物的一小部分，以制备甘油股票。甘油保存在-80℃冰箱中。 </li><li>使用证实，最高产细胞股票，开始独立的生产文化，并最终上清液2倍体积乙酸乙酯提取。使用真空蒸发系统，干燥提取物，并重新悬浮于100μl甲醇。转移到无菌的滤声器盘（〜1/4英寸直径）的提取物，并允许磁盘以干燥。此外，文化B.枯草芽孢杆菌在LB液体过夜。加入20μl的B.枯草芽孢杆菌培养至20毫升液体LB琼脂在45℃下的琼脂板，并允许巩固。将过滤器的磁盘上的板和孵育在37℃下（ 图7）。可替换地，该提取物可以被添加到液体培养内量化的活性，使用读板器（ 图7）的96孔板中。 </li></ol>

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没有利益冲突的声明。

异源生物合成中的关键步骤是在每个点的过程的三个程序：1）基因转移2）生物合成的重建，以及3）产品的分析中遇到。在任何阶段的问题，将破坏建立异源生物合成的最终目标。也许是最具挑战性的方面建立重组生物合成的过程，因为这是绝对必要的，让成功的分析。然而，重组是依赖于精心的设计和负责最终合成的遗传物质的转移。同样地，建立，灵敏的分析方法（如LC-MS）可以检测到小的?...

红霉素A是由革兰氏阳性土壤细菌糖多孢菌红霉素产生聚酮化合物的抗生素，和目前的生产已增量通过几十年的传统的诱变和筛选协议和最近通过过程优化方案1-6〜10 g / L的改善。诱变和筛选策略是常见的抗生素天然产品开发的困难，因此在培养和/或基因操纵本地生产主机，因为现成的抗菌活性或改进的的增长表型，以帮助选择。红霉素A的情况下，S.红霉素是一个缓慢的增长模式缺乏更直接的遗传操作技术（生物如大肠杆菌 ）的限制，因此，阻碍了生产的生物合成新的衍生产品的迅速改善。经确认的生产问题和解锁diversificati化合物，如红霉素A的可能性，研究界开始追求的理念异源生物合成（ 图1）7。这些努力的同时提供序列信息的红霉素A基因簇8-11。应当强调的是，序列复杂的天然产物的基因簇的数目已大大扩展12-16，提供的动力继续努力，在异源生物合成访问编码药用潜力。要做到这一点，异源的重建需要的新的主机满足的特定的生物合成途径的需要。E.大肠杆菌提供技术方便，一个广泛的分子生物学技术的跨越式组，代谢和制程工艺的产品发展战略。然而，相对于本地生产主机，E.大肠杆菌不表现出复杂的天然产物的生产相同的水平。因此，未知是否Ë。大肠杆菌可以作为一个可行的复杂的天然产物生物合成的异源的选择。然而，它被假定， 大肠杆菌大肠杆菌是一个理想的宿主生物体，可以实现异源生物合成。 有了这个共同的目标，最初的努力开始生产聚酮苷元6的deoxyerythronolide的B（6dEB）通过E.大肠杆菌 。但是，本机E.大肠杆菌代谢不能提供相当水平的丙酰辅酶A，和（2S） -甲基丙酰辅酶A需要支持6dEB生物合成的前体，也可以在新主机翻译后修改deoxyerythronolide的乙合酶（DEBS）酶。为了解决这些问题，由本地和外源酶的代谢途径是内置到E.大肠杆菌等，外生馈丙酸转化细胞内丙酰辅酶A，然后（2S） -甲基丙酰辅酶A，在工程完成这一途径，SFP基因放入第E染色体E.大肠杆菌 BL21（DE3）中，以产生一个新的菌株称为BAP1。的SFP酶是一个phosphopantetheinyl的的转移能够连接的4&#39;-phosphopantetheine上的辅助因子DEBS酶17,18。 DEBS基因（每个〜10 KB），然后放置在两个独立的可选诱导的T7启动子的表达载体。诱导后的温度（〜22℃）的一个关键调整后，DEBS基因协调表示内BAP1在启用状态中，能够产生6dEB 19。 追求全红霉素生物合成，然后开始使用类似的基因簇孢megalomicea或从S.的基因组成的混合途径红霉素，S.弗氏和S. venezuelae生产的中间体红霉素C和ð6 deoxyerythromycin，分别为20日至22日 。最近，我们扩展了这些努力，生产erythrom通过E.霉素A（红霉素）的临床相关的形式大肠杆菌 。在以前的工作中，我们的战略协调原来的20 S.红霉素的基因所需的聚酮化合物的生物合成，deoxysugar生物合成和附件，额外的剪裁，自电阻（ 图2）。总体而言，26（本地和外源）基因被设计为允许E.大肠杆菌产生红霉素A在4毫克/升23,24。这一结果建立了完整的生产一个复杂的聚酮的天然产物E.大肠杆菌和服务为基础，以利用这个新的生产选项，或寻求新的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> PCR仪</td><td> Eppendorf公司</td><td>的Mastercycler个人</td><td></td></tr><tr><td>二甲亚砜（DMSO） </td><td>费舍尔</td><td> BP231 </td><td></td></tr><tr><td>电穿孔</td><td> BioRad公司</td><td> Micropulser </td><td></td></tr><tr><td> IPTG </td><td>费舍尔</td><td> BP1620 </td><td></td></tr><tr><td>丙酸钠</td><td>西格玛</td><td> P1880 </td><td></td></tr><tr><td> L-阿拉伯糖</td><td>西格玛</td><td> A3256 </td><td></td></tr><tr><td>冷冻振荡器</td><td> Thermo Scientific的</td><td> MAXQ 4000 </td><td></td></tr><tr><td>微型离心</td><td> Ëppendorf </td><td>离心5415D </td><td></td></tr><tr><td> pGro7 </td><td>宝</td><td>伴侣质粒集（3340） </td><td></td></tr><tr><td> pET21，pET28，PCDF的二重奏-1 </td><td> EMD化学品</td><td> 69742-3，69864，71340 </td><td></td></tr><tr><td> LC-MS </td><td>美国应用生物系统公司</td><td> 3200 Q-陷阱</td><td></td></tr><tr><td>乙酸乙酯</td><td>西格玛</td><td> 270989 </td><td></td></tr><tr><td>甲醇</td><td>西格玛</td><td> 322415 </td><td></td></tr><tr><td>真空离心机</td><td> Eppendorf公司</td><td>集中5301 </td><td></td></tr><tr><td>旋转蒸发仪</td><td>步琪</td><td> R-200 </td><td></td></tr></tbody></table>

the logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin a produced through e. coli

复杂的天然产物的异源生产的是一种方法，旨在解决目前的局限性和未来的可能性。这是特别有用的那些化合物具有治疗价值，但不能充分地生产或将受益于生产一种改进形式的。实验过程可以细分成三个部分：1）基因转移; 2）异源重组; 3）产品分析。每个实验的成分是在不断优化，迎接挑战和预测的机会与这个新兴的方法。 异源生物合成的基因序列的识别珍贵的天然产品开始。此序列传送到一个​​异源宿主是复杂的生物合成途径的复杂性负责产物的形成。抗生素红霉素A是一个很好的例子。二十基因（最后的合成，共计50 KB）所需要的。此外，这些基因编码megasynthases的，域多酶〜300 kDa的大小。这种遗传材料必须被设计，并转移到大肠杆菌大肠杆菌重组生物合成。 PCR分离，操纵子建设，多cystronic的质粒，和电变换的使用将在下文中转移遗传聚类的红霉素A 至 E。 大肠杆菌 。 一旦转移，E.大肠杆菌细胞必须支持最终合成。这个过程也是充满挑战的巨大差异之间E.大肠杆菌和最原始的主机，负责复杂的天然产物的生成。该单元必须提供必要的支持生物合成的底物，并协调转移的基因簇表达活性酶。红霉素A，E 的情况下，在大肠杆菌细胞被设计，以提供两个前体（公关opionyl-CoA和（2S） - 甲基丙-CoA）的生物合成所需的。此外，基因序列的修改，质粒拷贝数，分子伴侣共表达，翻译后的酶修饰，和过程温度，也可以进行最后的红霉素A的形成。 最后，成功的生产必须进行评估。红霉素A的情况下，我们将介绍这两种方法。第一个是液相色谱 - 质谱（LC-MS），以确认和量化生产。也将红霉素A的生物活性的生物测定中， 枯草芽孢杆菌（Bacillus subtilis）的抗菌活性的测试是针对通过使用确认。评估分析，建立红霉素生物合成E.大肠杆菌 ，并设置为未来的工程努力，以改善或生产多样化和使用此方法的新的复杂的天然化合物的生产阶段。

这种方法所期望的结果是完全具有生物活性的天然产物生产的E.大肠杆菌异源宿主。这是最好的表示的LC-MS的结果，用于确认和量化生产（ 图6）和用于生物测定，以确认最后的活动（ 图7）的抗菌。在整体方案中的异源生物合成，这样的结果来定义成功。一旦完成，研究工作转向优化（无论是在细胞和工艺尺度）与分子工程。的最终目标包括原始化合物在异源系统?...

Watch this Scientific Journal Video about The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. 

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

通过异源生物合成红霉素A<em&gt; E.大肠杆菌</em包括下列实验步骤：1）基因转移; 2）异源重建;和3）产品分析。在生产的天然产物药物的使用，每个步骤进行说明的上下文中的动机，电势，和挑战<em&gt; E.大肠杆菌</em作为代理主机。

的逻辑，实验步骤和外源天然产物生物合成的潜力，拥有复杂的抗生素红霉素通过<em&gt; E.大肠杆菌</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

the-logic-experimental-steps-potential-heterologous-natural-product

Research

JoVE Journal

Biology

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

18.4K 次观看.  State University of New York at Buffalo. 通过异源生物合成红霉素A E.大肠杆菌

视频: The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

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. ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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

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

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

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

膜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 ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

人类诺沃克病毒凸，域的生产<em&gt;电子。大肠杆菌</em&gt; X射线晶体学

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

诱导位点特异性复制遇阻<i&gt;电子。大肠杆菌</i&gt;用荧光阻遏操作员系统

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...