这项工作是支持的，由惠康项目资助（078043/Z/05/Z）​​ROJW

第一部分:高通量增长的细菌培养。 1。在2毫升的自身诱导中过夜培养细菌利用24孔板<ol><li>微波灭菌的24孔板。 </li><li> ，接种1.5毫升新鲜生长的细菌菌落或冷冻甘油股票的的自身诱导介质（隔夜快件中）。我们通常接种三口井每突变体与三个独立克隆的殖民地。储备六口井为阳性和阴性对照。对于阳性对照，我们长大3野生型克隆和阴性对照，我们长大2已转化的非表达质粒的克隆。检查是否有足够的消毒板留下一个空白中只控制。 </li><li>成长的细胞在37℃下18小时，并以250rpm摇动。我们使用lac启动诱导BL21（DE3）中罗塞塔2细胞。自身诱导培养基中含有的葡萄糖和乳糖的混合物。这种细菌最初喂葡萄糖和然后开始使用乳糖，也诱导重组蛋白的表达"。 </li><li>取下盖子，并把机器人平台上的板。 </li></ol> PART B:机器人纯化重组蛋白。 2。准备的机器人平台<ol><li>化妆由20mM咪唑的洗涤缓冲液，0.1％的Triton X-100，0.5 M氯化钠，20mM的Tris-乙酸盐，pH值为7.9的10mM MgOAc 2，0.7 mM的ZnOAc 2，10％甘油，和洗脱缓冲液的组成的0.5M咪唑，0.1％的Triton X-100，0.5 M的NaCl，20mM的Tris-乙酸盐，pH值为7.9的10mM MgOAc 2，0.7 mM的ZnOAc 2，10％甘油。这些缓冲器将被用来洗珠后的标签的蛋白质已被绑定到这些，或分别从珠，洗脱蛋白质。 </li><li>化妆的细菌稀释液（100毫升蒸馏水中，用15μl的防沫试剂）。这种解决方案将用于使稀释过夜培养的细菌的光密度（A600）测量。消泡剂的存在下在稀释剂中防止形成气泡，会干扰与板读数器测量。 </li><li>裂解液的10倍快攻试剂，2微升lysonase每个样品15毫米MgOAc 2的 。快攻包含打破细菌细胞壁和促进细胞内的蛋白质释放的去垢剂和它的盐的组合。 lysonase是一个专有的混合溶菌酶和核酸。溶菌酶协助破坏细胞壁和核酸酶消化释放的细菌的核酸。 </li><li>可选:6M的盐酸胍解决方案。一些重组蛋白往往坚持的特氟纶涂层的移液针。盐酸胍溶液变性蛋白质和洗它们逐出更有效地比常规使用的水冲洗可水洗机器人吸头后，每个第一EP。 </li><li>准备的BCA蛋白定量试剂（二辛可宁酸）通过混合二辛可宁酸溶液（试剂A）和硫酸铜溶液（试剂B）:肽债券减少铜2 +从CuSO 4的成分中存在的BCA试剂为Cu 1 +由此的Cu 1 +的量是溶液中存在的肽键的数目成正比。在第二步骤中，两分子的二辛可宁酸螯合铜1 +在562 nm处的吸光度移位所导致，导致在一个紫色。的显色反应是时间依赖性的，并且通常需要几个小时的时间是确定的。在那之后的颜色是稳定的几个小时。 </li><li>大小合适的填充槽或板，并将其放置在他们的机器人平台的预分配的位置上。 </li><li>放置一个24孔板上的盐酸胍废物，两个明显的96 -孔板的OD 600和吸光度测量和一个蓝色的第96 -孔板Ê自己的立场纯化的蛋白的平台上。将一个96深孔板的磁性底座上。 </li><li>稀释MagneHis镍颗粒结合蛋白通过形成螯合物与他们的His-标签在蒸馏水中的5倍，并填充到胎圈搅拌单元。接通搅拌器保持在悬浮液中的小珠。 </li><li>切换机器人平台上，并冲洗移液针几分钟，以除去气泡，否则会干扰移液精度。可重复使用的移液的针被刷新个人之间的移液操作步骤。可替换地，可以使用一次性吸头。所有随后的步骤进行机器人。该机器人的协议可应要求提供。 </li></ol> 3。经过测量OD600细胞生长<ol><li> 10微升过夜培养物稀释在90微升稀释剂溶液。 </li><li>测量的OD 600，以保证细菌生长在相似密度（ 图1）。 </li></ol> 4。磁珠结合的细胞破坏释放的蛋白质，并允许<ol><li>磁性镍微珠悬浮液分配到第二个24孔板的各孔中加入100μl。 </li><li>每个小规模的细菌培养的900微升转移到24孔板上，含有小珠。添加100μl的的10倍快攻/ lysonase的混合。 </li><li>快速摇动（每分钟800转），在室温下的30分钟的振动平台转移到24孔板上。细菌细胞壁被破坏由一个组合的机械力和化学作用的重组产生的蛋白被释放到溶液中。凭借其亲和标签，他们立即绑定顺的螯合镍珠。 </li></ol> 5。珠洗水<ol><li>包含重新悬浮磁珠的细胞溶胞产物转移到在96深孔板，其被定位在支架上与magnetic棒，井间的滑动。 96 - 孔板有一个正方形的锥形底部，允许更容易地除去上清液。该井可容纳体积达至2.1毫升，但都充满了更小的体积。这使我们能够进行剧烈振荡的步骤没有样品交叉污染的情况下飞溅。 </li><li>吸头进行清洗6 M盐酸胍个人之间的步移液操作。这一步是至关重要的净化TFIIB和其他的"粘性"的蛋白质，以避免交叉污染。与其他蛋白质，它可能是足够广泛冲洗针与水之间的移液管的步骤。 </li><li>磁性支架的磁性杆，吸引的顺磁性珠，拉它们远离井的中心，并允许移液针免费访问上清液。弃去上清液。 </li><li>第一次添加到500微升的洗涤缓冲液的24孔板上，并随后将其转移到96孔板中以电子邮件nsure珠粒的完全转移。的24孔板上，从摇动器中除去。 </li><li>另外500微升的洗涤缓冲液中直接加入到96孔板中，该板被转移到振动筛，并剧烈振摇1分钟。该板向后移动到磁性底座和丢弃的洗涤缓冲液。 </li><li>此步骤重复两次，并在洗涤过程完成后通过除去任何缓冲从板残余。 </li></ol> 6。洗脱洗脱缓冲液中蛋白质<ol><li> 100微升洗脱缓冲液添加到珠粒，该板被移动到振动筛，并剧烈振摇30分钟，在室温下。 </li><li>该板向后移动到振动筛和洗脱液，含有纯化的重组蛋白，被转移到一个新的平板。 </li></ol> 7。测量浓度的纯化蛋白<ol><li> 190微升的BCA试剂混合的一个明确的96孔板中，并转移到10微升的亲蛋白质的解决方案。 </li><li>经过几个小时的孵育（通常为5-6或过夜），测量的吸光度和BSA标准进行比较，以确定每个纯化的蛋白制剂（图2）的浓度。 </li><li>在适当的浓度（图3），将纯化的蛋白质，现在可以用于下游应用。 </li></ol> 8。代表性的成果的纯化的协议提供了两个质量控制阶段，显示了其中的例子。我们能够识别并记录可能出现的问题，在细菌的生长阶段（ 图1），后来在评估的纯化蛋白的产量（ 图2）。我们通常蛋白质纯化，并对其进行测试，一式三份。结合的质量控制措施，这给了我们信心，我们的功能分析，我们观察到任何变化的突变是由于phenotypE（ 图3），并且不是由于实验的变化的或失败的纯化。获得的收益率通常范围从50-200微克，超过足够的各种功能检测。 <img alt="图1" src="/files/ftp_upload/4110/4110fig1.jpg" /> 图1。直方图与过夜培养的24孔板的OD测量六个TFIIB突变的变体，以及野生型TFIIB三个克隆已生长。只携带非表达质粒的构建和一个与介质的两个克隆作为阴性对照。 OD测量结果表明，在增长率之间的文化，也有小的变化。 <img alt="图2" src="/files/ftp_upload/4110/4110fig2.jpg" /> 图2。从这些培养获得的蛋白质产量的BCA法所确定的，并通过SDS-PAGE证实。不表达变体之一的是，在高的水平。在COMbination与图1中 ，我们可以得出这样的结论:这是由于差细胞生长，但由于蛋白的表达不正确所引起的。 <img alt="图3" src="/files/ftp_upload/4110/4110fig3.jpg" /> 图3。的转录测定的代表性的结果 。 RNAP小流产的成绩单，生产上，我们测得的刺激活动的TFIIB的变种。在这里，示出一个完整的图书馆TFIIB残渣K87的单个氨基酸取代的刺激效应。高度的再现性确认由小的错误率。描述了三个突变体的表现相比，野生型（WT），阴性对照组（NC）和洗脱缓冲液的样本凝胶只控制下。

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没有利益冲突的声明。

这里所描述的自动重组蛋白的纯化方法允许在高重现性的条件下在一个小规模的格式的大量的突变体蛋白的生产和纯化，以最小的人为干预。 图1和图2示出的结果，系统的质量控制和示例纯化的蛋白质。 图3表明，在本实施例中使用的纯化的转录因子在一个高度可重复的方式在功能测定执行。 即使为古细菌TFIIB纯化程序开发，它是广泛适用的亲和标签的蛋白的纯化。因此这种自动净化协议的使用将显着促进重组蛋白的生化分析，从而将进一步了解我们的蛋白质 - 蛋白质相互作用的规模是难以实现手动。

<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>隔夜快递即时TB中</td><td>默克化工有限公司</td><td> 71491-4 </td><td></td></tr><tr><td>快攻</td><td> Promega公司有限公司</td><td> V8573 </td><td></td></tr><tr><td> Lysonase生物处理试剂/ 1毫升</td><td>默克化工有限公司</td><td> 71230 </td><td></td></tr><tr><td>消泡剂204 </td><td> Sigma-Aldrich公司有限责任公司</td><td> A6426 </td><td></td></tr><tr><td> MagneHis镍颗粒</td><td> Promega公司</td><td> V8565 </td><td></td></tr><tr><td>咪唑</td><td> Sigma-Aldrich公司有限责任公司</td><td> 56750 </td><td></td></tr><tr><td> Trizma碱</td><td> Sigma-Aldrich公司比较任何公司</td><td> 93362 </td><td></td></tr><tr><td>氯化钠</td><td> VWR </td><td> 27810.295 </td><td></td></tr><tr><td>二喹啉甲酸蛋白测定</td><td> Sigma-Aldrich公司有限责任公司</td><td> BCA1-1KT </td><td></td></tr><tr><td>深孔板2.2毫升广场井PP PK10 </td><td> Anachem有限公司</td><td> 1810至1800年</td><td></td></tr><tr><td>微孔板微孔板96孔平底聚苯乙烯治疗12的衣袖，5板清晰0.4毫升以及体积128毫米毫米x 86毫米的Thermo Scientific Nunc公司</td><td>默飞世尔科技有限公司</td><td> DIS-984-090M </td><td></td></tr><tr><td>微孔板蓝</td><td> VWR </td><td> NUNC367001 </td><td></td></tr><tr><td> 24井区块RB（24） </td><td> Qiagen公司</td><td> 19583 </td><td></td></tr><tr><td>盐酸胍</td><td> VWR </td><td> ALFAA13543.0B </td><td></td></tr><tr><td>水洗针TheOnyx </td><td> Aviso有限公司</td><td> 8152-317001 </td><td></td></tr><tr><td>磁珠试剂架</td><td> Aviso有限公司</td><td> 8152-035003 </td><td></td></tr><tr><td>酶标仪协同HT </td><td>百特</td><td> 4200-000043 </td><td></td></tr><tr><td>机器人平台TheOnyx 44OH/150/100 </td><td> Aviso有限公司</td><td> 8145-050046 </td><td></td></tr><tr><td>微孔板振荡器Variomag Teleshaker </td><td> Inheco </td><td> 3800047 </td><td></td></tr><tr><td> 96孔磁铁A型</td><td> Qiagen公司</td><td> 36915 </td><td></td></tr></tbody></table>

high-throughput purification of affinity-tagged recombinant proteins

X-射线晶体给出的方法是用于获得蛋白质的结构的详细视图。这样的研究需要辅之以进一步的生化分析，以获得详细的结构/功能关系的深入了解。寡核苷酸基因合成技术的进展进行大规模突变的战略越来越可行的，其中包括所有其他19个氨基酸的替代目标残​​留物。增益或损失的函数的表型，然后使系统的得出的结论，如特定的残基的催化活性，蛋白质的稳定性和/或蛋白质 - 蛋白质相互作用的特异性的贡献。 以属性的不同表型的突变的性质 - 而不是波动的实验条件下 - 这是至关重要的纯化和分析的蛋白质在一个控制和可重复的方式。高吞吐量的战略和自动化的手动协议，机器人液体处理平台，创造了机会进行这样的复杂的分子生物学方法很少人工干预，最少的错误率1-5。 这里，我们提出的带有His标记的重组蛋白中的高通量的方式纯化的一般方法。在最近的研究中，我们采用这种方法的详细调查TFIIB，结构与功能的基础转录机器的一个组成部分。 TFIIB是不可缺少的启动子体外转录前起始复合物6-8成的RNA聚合酶的招聘是必不可少的。 TFIIB包含柔性连接域贯通RNA聚合酶9-11裂的活性部位。这种链接域赋予两个生化量化活动TFIIB，即（i）在"流产"的转录起始阶段刺激的催化活性，及（ii）额外的贡献，具体招聘的RNA聚合酶的前起始复合4,5,12。我们利用高通量纯化的方法来产生单人间，双人间和三人间替代和缺失突变在TFIIB连接器和，随后分析他们的功能分析的刺激效应对催化活性的RNA聚合酶4。总之，我们产生，纯化和分析381突变体 - 一个将是费时和费力的手动执行的任务。我们在生产和检测中的蛋白质multiplicates，让我们体会到任何实验性的变化，给了我们一个清晰的思路，我们的研究结果的可重复性。 此方法作为一个通用的协议，用于带有His标记的蛋白质纯化，并已成功地用于纯化其它重组蛋白。目前优化的24种蛋白质的纯化，但也可以适于净化多达96个蛋白质。

Watch this Scientific Journal Video about High-throughput Purification of Affinity-tagged Recombinant Proteins at JoVE.com

High-throughput Purification of Affinity-tagged Recombinant Proteins

我们描述了一种方法，亲和标签的重组蛋白的纯化，使用的液体处理机器人。此方法通常是适用于小规模纯化的可溶性的带有His标记的蛋白质在高吞吐量的格式。

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

high-throughput-purification-of-affinity-tagged-recombinant-proteins

Nanyang Technological University

Research

JoVE Journal

Biology

14.1K 次观看.  Imperial College London. 我们描述了一种方法，亲和标签的重组蛋白的纯化，使用的液体处理机器人。此方法通常是适用于小规模纯化的可溶性的带有His标记的蛋白质在高吞吐量的格式。

视频: High-throughput Purification of Affinity-tagged Recombinant Proteins

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

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

在甲醇酵母表达的重组蛋白毕赤酵母</em

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

使用油脂全线Bicelle方法的膜蛋白的高通量结晶

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

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 ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

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 ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...